Coumarin compounds and their uses as fluorescent labels

ABSTRACT

The present application relates to new coumarin compounds and their uses as fluorescent labels. The compounds may be used as fluorescent labels for nucleotides in nucleic acid sequencing applications.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/738,560, filed Jan. 9, 2020 and to be issued as U.S. Pat. No.10,907,196, which is a continuation of U.S. application Ser. No.16/282,783, filed Feb. 22, 2019 and now U.S. Pat. No. 10,533,211, whichis a continuation of U.S. application Ser. No. 15/851,014, filed Dec.21, 2017 and now U.S. Pat. No. 10,214,768, which claims the benefit ofpriority to U.S. Provisional Application No. 62/438,006, filed Dec. 22,2016, each of which is incorporated by reference in its entirety.

BACKGROUND Field

The present application relates to coumarin compounds. The compounds maybe used as fluorescent labels, particularly for nucleotide labeling innucleic acid sequencing applications.

Background

Non-radioactive detection of nucleic acids utilizing fluorescent labelsis an important technology in molecular biology. Many proceduresemployed in recombinant DNA technology previously relied on the use ofnucleotides or polynucleotides radioactively labeled with, for example³²P. Radioactive compounds permit sensitive detection of nucleic acidsand other molecules of interest. However, there are serious limitationsin the use of radioactive isotopes such as their expense, limited shelflife and more importantly safety considerations Eliminating the need forradioactive labels enhances safety whilst reducing the environmentalimpact and costs associated with, for example, reagent disposal. Methodsamenable to non-radioactive fluorescent detection include by way ofnon-limiting example, automated DNA sequencing, hybridization methods,real-time detection of polymerase-chain-reaction products andimmunoassays.

For many applications it is desirable to employ multiple spectrallydistinguishable fluorescent labels in order to achieve independentdetection of a plurality of spatially overlapping analytes. In suchmultiplex methods the number of reaction vessels may be reduced tosimplify experimental protocols and facilitate the production ofapplication-specific reagent kits. In multi-color automated DNAsequencing systems for example, multiplex fluorescent detection allowsfor the analysis of several different nucleotide bases in a singleelectrophoresis lane. This increases throughput over detection systemsusing a single-color, and also can reduce the uncertainties associatedwith inter-lane electrophoretic mobility variations.

However, multiplex fluorescent detection can be problematic and thereare a number of important factors, which constrain selection offluorescent labels. First, it is difficult to find dye compounds whoseabsorption and emission spectra are suitably spectrally resolved. Inaddition, when several fluorescent dyes are used together, simultaneousexcitation may be difficult because the absorption bands of the dyes fordifferent spectral regions may be widely separated. Many excitationmethods use high power lasers and therefore the dye must have sufficientphoto-stability to withstand such laser excitation. A finalconsideration of particular importance in molecular biology methods isthat the fluorescent dyes must be compatible with the reagentchemistries. Thus, for example the fluorescent dyes used in DNAsynthesis or sequencing reactions must be compatible with the solventsand reagents, buffers, polymerase enzymes and ligase enzymes used inthose reactions. In one example, PCT Publication No. WO 2007/135368describes a class of rhodamine compounds used as fluorescent labels.

Coumarin dyes family has attracted attention of chemists due to theirremarkable spectral properties. Nevertheless, there are only a fewphoto-stable fluorescent dyes with large Stokes shifts (LSS) that arecommercially available. Most of these dyes also contain the coumarinfragment as a scaffold. For example, most of the dyes from Dyomics arecoumarin derivatives absorbing at about 480-520 nm, and emitting in theregion of 560-630. Other examples of this class of coumarin dyes includephosphorylated coumarin based dyes as disclosed in U.S. Publication No.2014/0220588 and commercially available dyes Star440SXP and Star 470SXPfrom Abberior. Another practically useful coumarin dye is AlexaFluor™430 with absorption and emission maxima at 434 nm and 539 nmrespectively. Other LSS fluorescent dyes include Pacific Orange™ (abs.390 nm, emission 540 nm; Stokes shift 150 nm, Invitrogen) and BDHorizon™ V500 (abs. 415 nm, emission 500 nm; Stokes shift 85 nm, BDBiosciences) Chromeo™ 494 (abs. 494 nm, emission 628 nm, Stokes Shift134 nm, Active Motive).

SUMMARY

Described herein are novel coumarin derivatives and their use asbio-molecule labels, particularly as labels for nucleotides used innucleic acid sequencing. When such dyes are used for the preparation ofbio-molecule conjugates, improvements can be seen in the length,intensity and quality of sequencing read obtainable due to the use ofthese new fluorescent compounds.

Some embodiments described herein are related to coumarin compounds ofFormula (I), salts or mesomeric forms thereof:

wherein R¹ is

and wherein R¹ is optionally substituted with one or more substituentsselected from the group consisting of alkyl, substituted alkyl, alkoxy,alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl,halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy,C-amido, N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonate,S-sulfonamido, N-sulfonamido, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted cycloalkyl, andoptionally substituted heterocyclyl;

each R², R³, R⁴, R⁵, and R⁹ is independently selected from the groupconsisting of H, alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl,haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano,hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido,N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido,N-sulfonamido, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl and optionallysubstituted heterocyclyl;

each R⁶, R^(10a), R^(10b) and R^(10c) is independently selected from thegroup consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl,aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfonyl hydroxide,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted carbocyclyl, and optionally substitutedheterocyclyl;

each R⁷ and R⁸ is independently selected from the group consisting of H,alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl,haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano, hydroxy,hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido,nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido,N-sulfonamido, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl and optionallysubstituted heterocyclyl;

alternatively, R⁶ and R⁷ together with the atoms to which they areattached form a ring or ring system selected from the group consistingof optionally substituted 5-10 membered heteroaryl or optionallysubstituted 5-10 membered heterocyclyl;

X is selected from the group consisting of O, S, NR¹¹, and Se;

R¹¹ is selected from the group consisting of H, alkyl, substitutedalkyl, alkenyl, alkynyl, aminoalkyl, carboxyalkyl, sulfonatoalkyl,haloalkyl, heteroalkyl, alkoxyalkyl, sulfo, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted carbocyclyl,and optionally substituted heterocyclyl; and

the bond represented by a solid and dashed line

is selected from the group consisting of a single bond and a doublebond, provided that when

is a double bond, then R³ is absent.

Some embodiments described herein are related to fluorescent compoundsof Formula (II) with a Stokes shift at least about 60 nm, salts, ormesomeric forms thereof:

wherein R^(Het) is a 5 to 10 membered heteroaryl optionally substitutedwith one or more R¹⁰;

each R¹, R², R³, R⁴, and R⁵ is independently selected from the groupconsisting of H, alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl,haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano,hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido,N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido,N-sulfonamido, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl and optionallysubstituted heterocyclyl;

R⁶ is selected from the group consisting of H, alkyl, substituted alkyl,alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl,sulfonyl hydroxide, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted carbocyclyl, and optionallysubstituted heterocyclyl;

each R⁷ and R⁸ is independently selected from the group consisting of H,alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl,haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano, hydroxy,hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido,nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido,N-sulfonamido, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl and optionallysubstituted heterocyclyl;

alternatively, R⁶ and R⁷ together with the atoms to which they areattached form a ring or ring system selected from the group consistingof optionally substituted 5-10 membered heteroaryl or optionallysubstituted 5-10 membered heterocyclyl;

each R¹⁰ is independently selected from the group consisting of alkyl,substituted alkyl, alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl,alkoxyalkyl, sulfonyl hydroxide, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted carbocyclyl, andoptionally substituted heterocyclyl;

the bond represented by a solid and dashed line

is selected from the group consisting of a single bond and a doublebond, provided that when

is a double bond, then R³ is absent.

Some embodiments described herein are related to nucleotide oroligonucleotide labeled with a compound of Formula (I) or Formula (II).

Some embodiments described herein are related to kits containing one ormore nucleotides where at least one nucleotide is a labeled nucleotidedescribed herein.

Some further embodiments described herein are related to methods ofsequencing including incorporating a labeled nucleotide described hereinin a sequencing assay, and detecting the labeled nucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the usability of the A-nucleotide labeled with thenew coumarin dye I-16 as described herein for sequencing analysis.

FIG. 2 illustrates the usability of the A-nucleotide labeled with acommercial fluorescent dye DY510XL with long Stokes shift for sequencinganalysis.

FIG. 3 illustrates the usability of the A-nucleotide labeled with acommercial fluorescent dye Chromeo494 with long Stokes shift forsequencing analysis.

DETAILED DESCRIPTION

Embodiments described herein relate to new coumarin dyes and theirderivatives of the structure of Formula (I) for use as fluorescentlabels. Further embodiments relate to fluorescent compound of thestructure of Formula (II) with a Stokes shift of at least about 60 nm.

These new fluorescent dyes may be used as fluorescent labels,particularly for nucleotide labeling in nucleic acid sequencingapplications. Methods of preparing these fluorescent dyes and downstreamsequencing applications utilizing these dyes are also exemplified.

Surprisingly, it has been discovered that the fluorescence intensitiesof the new dyes and their bio-conjugates are nearly equal whenirradiated with either blue or green light sources. For example, whenthe dyes are excited with 460 nm (blue) and 540 nm (green) laser or LED,the fluorescence intensities are about the same in some cases. Asdescribed below, this property holds true in solution and on flow cells,enabling simplified sequencing analysis with high quality.

Compounds of Formula (I)

Some embodiments described herein are related to new coumarinderivatives of Formula (I), or salts, mesomeric forms thereof:

wherein R¹ is

and wherein R¹ is optionally substituted with one or more substituentsselected from the group consisting of alkyl, substituted alkyl, alkoxy,alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl,halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy,C-amido, N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonate,S-sulfonamido, N-sulfonamido, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted cycloalkyl, andoptionally substituted heterocyclyl;

each R², R³, R⁴, R⁵, and R⁹ is independently selected from the groupconsisting of H, alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl,haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano,hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido,N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido,N-sulfonamido, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl and optionallysubstituted heterocyclyl;

each R⁶, R^(10a), R^(10b) and R^(10c) is independently selected from thegroup consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl,aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfonyl hydroxide,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted carbocyclyl, and optionally substitutedheterocyclyl;

each R⁷ and R⁸ is independently selected from the group consisting of H,alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl,haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano, hydroxy,hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido,nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido,N-sulfonamido, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl and optionallysubstituted heterocyclyl;

alternatively, R⁶ and R⁷ together with the atoms to which they areattached form a ring or ring system selected from the group consistingof optionally substituted 5-10 membered heteroaryl or optionallysubstituted 5-10 membered heterocyclyl;

X is selected from the group consisting of O, S, NR¹¹, and Se;

R¹¹ is selected from the group consisting of H, alkyl, substitutedalkyl, alkenyl, alkynyl, aminoalkyl, carboxyalkyl, sulfonatoalkyl,haloalkyl, heteroalkyl, alkoxyalkyl, sulfo, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted carbocyclyl,and optionally substituted heterocyclyl; and

the bond represented by a solid and dashed line

is selected from the group consisting of a single bond and a doublebond, provided that when

is a double bond, then R³ is absent.

In some embodiments of the compounds of Formula (I), the alkyl orsubstituted alkyl disclosed herein is C₁₋₁₂ alkyl, or more preferablyC₁₋₆ alkyl. In some embodiments, the alkoxy disclosed herein is C₁₋₁₂alkoxy, or more preferably C₁₋₆ alkoxy. In some embodiments, the alkenyland alkynyl groups disclosed herein are C₂₋₆ alkenyl and C₂₋₆ alkynyl.In some embodiments, the haloalkyl, haloalkoxy, aminoalkyl,hydroxyalkyl, heteroalkyl groups disclosed herein are C₁₋₁₂ haloalkyl,C₁₋₁₂ haloalkoxy, C₁₋₁₂ aminoalkyl, C₁₋₁₂ hydroxyalkyl and C₁₋₁₂heteroalkyl; more preferably C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, C₁₋₆aminoalkyl, C₁₋₆ hydroxyalkyl and C₁₋₆ heteroalkyl. In some embodiments,the alkoxyalkyl group disclosed herein is C₁₋₆ alkoxy(C₁₋₆ alkyl). Insome embodiments, the optionally substituted aryl disclosed herein isoptionally substituted C₆₋₁₀ aryl, for example, phenyl. In someembodiments, the optionally substituted heteroaryl disclosed herein isoptionally substituted 5-10 membered heteroaryl; more preferably,optionally substituted 5-6 membered heteroaryl. In some embodiments, theoptionally substituted carbocyclyl disclosed herein is optionallysubstituted 3-7 membered carbocyclyl, in particular 3-7 memberedcycloalkyl. In some embodiments, optionally substituted heterocyclyldisclosed herein are optionally substituted 3-7 membered heterocyclyl,more preferably 5-6 membered heterocyclyl.

In some embodiments of the compounds of Formula (I), any of R² throughR⁹ may be selected from an alkyl substituted with one or moresubstituents selected from carboxyl (—CO₂H) or carboxylate (CO₂ ⁻),sulfo (SO₃H) or sulfonate (SO₃ ⁻) groups. In some such embodiments, thecompounds of Formula (I) are also represented by its salt form Formula(I′) with an organic or inorganic cation:

wherein K is an organic or inorganic cation, and n is an integerselected from 1 to 20.

In some embodiments of the compounds of Formula (I) or (I′), R¹ is

In some such embodiments, X is O. In some other embodiments, X is O. Insome such embodiments, the compounds of Formula (I) are also representedby Formula (Ia):

In some embodiments, R¹ is substituted with one or more substituentsselected from the group consisting of alkyl, halo, and C-carboxy. In oneembodiment, R¹ is substituted with a chloro (i.e., —Cl). In anotherembodiment, R¹ is substituted with a carboxyl (i.e., —C(O)OH).

In some embodiments of the compounds of Formula (I) or (I′), R¹ is

In some other embodiments, R¹ is

It is understood that when R^(10a), R^(10b) or R^(10c) is connected to apyridyl group bearing a positive charge on the nitrogen atom, R^(10a),R^(10b) or R^(10c) may contain a negative charge so that R¹ as a wholeis charge neutral. Alternatively, when R^(10a), R^(10b) or R^(10c) isconnected to a pyridyl group bearing a positive charge on the nitrogenatom, the compound described herein may contain a counterion so that thecompound as a whole is charge neutral. In some such embodiments,R^(10a), R^(10b) or R^(10c) is a substituted alkyl, for example,substituted C₁, C₂, C₃, C₄, C₅, or C₆ alkyl. In some such embodiment,the alkyl is substituted with carboxyl (—CO₂H), carboxylate (CO₂ ⁻),sulfo (SO₃H), or sulfonate (SO₃ ⁻). In some such embodiments, thecompounds of Formula (I) are also represented by Formula (Ib) and (Ic)or their salt Formula (Ib′) and (Ic′):

wherein Y is an anion that is capable of forming a charge neutralcompound with Ib. In some embodiment, Y is an anion derived from organicor inorganic acid. In some embodiments, Y is a halogen anion.

In some embodiments of the compounds of Formula (I), (I′), (Ia), (Ib),(Ib′), (Ic), or (Ic′), the bond represented by a solid and dashed line

is a double bond and the compounds are also presented by Formula (I-1),(I′-1), (Ia-1), (Ib-1), (Ib′-1), (Ic-1), or (Ic′-1):

In some embodiments of the compounds of Formula (I), (I-1), (I′-1),(Ia-1), (Ib-1), (Ib′-1), (Ic-1), or (Ic′-1), R² is alkyl. In oneembodiment, R² is methyl. In some other embodiments, R² is H.

In some embodiments of the compounds of Formula (I), (I-1), (I′-1),(Ia-1), (Ib-1), (Ib′-1), (Ic-1), or (Ic′-1), at least one of R⁴ and R⁵is alkyl. In some such embodiments, each R⁴ and R⁵ is alkyl. In oneembodiment, both R⁴ and R⁵ are methyl. In some alternative embodiments,at least one of R⁴ and R⁵ is H. In one such embodiment, both R⁴ and R⁵are H.

In some embodiments of the compounds of Formula (I), (I′), (Ia), (Ib) or(Ib′), the bond represented by a solid and dashed line

is a single bond and the compounds are also presented by Formula (I-2),(I′-2), (Ia-2), (Ib-2), (Ib′-2), (Ic-2), or (Ic′-2):

In some embodiments of the compounds of Formula (I), (I-2), (I′-2),(Ia-2), (Ib-2), (Ib′-2), (Ic-2), or (Ic′-2), at least one of R² and R³is alkyl. In some further embodiments, both R² and R³ are alkyl. In oneembodiment, both R² and R³ are methyl. In some other embodiments, atleast one of R² and R³ is H. In one embodiment, both R² and R³ are H.

In some embodiments of the compounds of Formula (I), (I-2), (I′-2),(Ia-2), (Ib-2), (Ib′-2), (Ic-2), or (Ic′-2), at least one of R⁴ and R⁵is H. In one such embodiment, both R⁴ and R⁵ are H. In some alternativeembodiments, at least one of R⁴ and R⁵ is alkyl. In some suchembodiments, each R⁴ and R⁵ is alkyl. In one embodiment, both R⁴ and R⁵are methyl.

In some embodiments of the compounds of Formula (I), (I′), (I-1),(I′-1), (Ia-1), (Ib-1), (Ib′-1), (Ic-1), (Ic′-1), (I-2), (I′-2), (Ia-2),(Ib-2), (Ib′-2), (Ic-2), or (Ic′-2), R⁶ is a substituted alkyl, forexample, substituted C₁, C₂, C₃, C₄, C₅, or C₆ alkyl. In one embodiment,R⁶ is alkyl substituted with carboxyl. In some embodiments, R⁶ is analkyl substituted with —C(O)OR¹², and wherein R¹² is selected from thegroup consisting of optionally substituted alkyl, optionally substitutedaryl, optionally substituted heteroaryl, and optionally substituted 3 to7 membered cycloalkyl. In one such embodiment, R¹² is an alkyl, forexample, methyl, ethyl, or t-butyl. In some further embodiments, R⁶ isan alkyl substituted with —C(O)NR¹³R¹⁴, and wherein each R¹³ and R¹⁴ isindependently selected from H, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted heteroaryl and optionallysubstituted 3 to 7 membered cycloalkyl. In some further embodiments, R¹³and R¹⁴ is independently selected from an alkyl substituted with one ormore substituents selected from the group consisting of carboxyl,carboxylate, —C(O)OR¹¹, sulfo and sulfonate.

In some embodiments of the compounds of Formula (I), (I′), (I-1),(I′-1), (Ia-1), (Ib-1), (Ib′-1), (Ic-1), (Ic′-1), (I-2), (I′-2), (Ia-2),(Ib-2), (Ib′-2), (Ic-2), or (Ic′-2), R⁷ is H.

In some alternative embodiments of the compounds of Formula (I), (I′),(I-1), (I′-1), (Ia-1), (Ib-1), (Ib′-1), (I-2), (I′-2), (Ia-2), (Ib-2),or (Ib′-2), R⁶ and R⁷ are joined together with the atoms to which theyare attached to form an optionally substituted 3 to 10 memberedheterocyclyl, for example, an optionally substituted 6 memberedheterocyclyl. In some such embodiments, the optionally substitutedheterocyclyl contains one heteroatom. In some such embodiments, theoptionally substituted heterocyclyl is substituted with one or morealkyls, for example, methyl.

In some embodiments of the compounds of Formula (I), (I′), (I-1),(I′-1), (Ia-1), (Ib-1), (Ib′-1), (Ic-1), (Ic′-1), (I-2), (I′-2), (Ia-2),(Ib-2), (Ib′-2), (Ic-2), or (Ic′-2), R⁸ is H.

In some embodiments of the compounds of Formula (I), (I′), (I-1),(I′-1), (Ia-1), (Ib-1), (Ib′-1), (Ic-1), (Ic′-1), (I-2), (I′-2), (Ia-2),(Ib-2), (Ib′-2), (Ic-2), or (Ic′-2), R⁹ is H.

In some specific embodiments, exemplary compounds of Formula (I) includeCompounds I-1 through I-20 and Compounds I-22 through I-32 as shown inTable 1 below:

TABLE 1 I-1 

I-2 

I-3 

I-4 

I-5 

I-6 

I-7 

I-11

I-12

I-13

I-14

I-15

I-16

I-17

I-8 

I-9 

I-10

I-22

I-24

I-26

I-18

I-19

I-20

I-23

I-25

I-27

I-28

I-30

I-32

I-29

I-31

In some embodiments of the compounds of Formula (I), the compound iscovalently attached to a nucleotide or oligonucleotide via R⁶, andwherein R⁶ is a substituted alkyl, for example, a substituted C₁, C₂,C₃, C₄, C₅, or C₆ alkyl. In one embodiment, R⁶ is an alkyl substitutedwith carboxyl.

In some alternative embodiments, the compound is covalently attached toa nucleotide or oligonucleotide via R⁸, and wherein R⁸ is a substitutedalkyl, for example, a substituted C₁, C₂, C₃, C₄, C₅, or C₆ alkyl. Inone embodiment, R⁸ is an alkyl substituted with carboxyl.

In some alternative embodiments, the compound is covalently attached toa nucleotide or oligonucleotide via R^(10a), R^(10b), or R^(10c), andwherein each of R^(10a), R^(10b), or R^(10c) is a substituted alkyl, forexample, a substituted C₁, C₂, C₃, C₄, C₅, or C₆ alkyl. In oneembodiment, each of R^(10a), R^(10b), or R^(10c) is an alkyl substitutedwith carboxyl.

In some embodiments, the structure of compound of Formula (I) isrepresented in one or more mesomeric forms:

Compounds of Formula (II)

Some embodiments described herein are related to fluorescent compoundsof Formula (II) with a Stokes shift at least about 60 nm, or salts,mesomeric forms thereof:

wherein R^(Het) is a 5 to 10 membered heteroaryl optionally substitutedwith one or more R¹⁰;

each R¹, R², R³, R⁴, and R⁵ is independently selected from the groupconsisting of H, alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl,haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano,hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido,N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido,N-sulfonamido, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl and optionallysubstituted heterocyclyl;

R⁶ is selected from the group consisting of H, alkyl, substituted alkyl,alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl,sulfonyl hydroxide, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted carbocyclyl, and optionallysubstituted heterocyclyl;

each R⁷ and R⁸ is independently selected from the group consisting of H,alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl,haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano, hydroxy,hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido,nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido,N-sulfonamido, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl and optionallysubstituted heterocyclyl;

alternatively, R⁶ and R⁷ together with the atoms to which they areattached form a ring or ring system selected from the group consistingof optionally substituted 5-10 membered heteroaryl or optionallysubstituted 5-10 membered heterocyclyl;

each R¹⁰ is independently selected from the group consisting of alkyl,substituted alkyl, alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl,alkoxyalkyl, sulfonyl hydroxide, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted carbocyclyl, andoptionally substituted heterocyclyl;

the bond represented by a solid and dashed line

is selected from the group consisting of a single bond and a doublebond, provided that when

is a double bond, then R³ is absent.

In some embodiments, the fluorescent compounds of Formula (II) have aStokes shift ranging from about 60 nm to about 100 nm, or from about 60nm to about 90 nm.

In some embodiments of the compounds of Formula (II), the alkyl orsubstituted alkyl disclosed herein is C₁₋₁₂ alkyl, or more preferablyC₁₋₆ alkyl. In some embodiments, the alkoxy disclosed herein is C₁₋₁₂alkoxy, or more preferably C₁₋₆ alkoxy. In some embodiments, the alkenyland alkynyl groups disclosed herein are C₂₋₆ alkenyl and C₂₋₆ alkynyl.In some embodiments, the haloalkyl, haloalkoxy, aminoalkyl,hydroxyalkyl, heteroalkyl groups disclosed herein are C₁₋₁₂ haloalkyl,C₁₋₁₂ haloalkoxy, C₁₋₁₂ aminoalkyl, C₁₋₁₂ hydroxyalkyl and C₁₋₁₂heteroalkyl; more preferably C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, C₁₋₆aminoalkyl, C₁₋₆ hydroxyalkyl and C₁₋₆ heteroalkyl. In some embodiments,the alkoxyalkyl group disclosed herein is C₁₋₆ alkoxy(C₁₋₆ alkyl). Insome embodiments, the optionally substituted aryl disclosed herein isoptionally substituted C₆₋₁₀ aryl, for example, phenyl. In someembodiments, the optionally substituted heteroaryl disclosed herein isoptionally substituted 5-10 membered heteroaryl; more preferably,optionally substituted 5-6 membered heteroaryl. In some embodiments, theoptionally substituted carbocyclyl disclosed herein is optionallysubstituted 3-7 membered carbocyclyl, in particular 3-7 memberedcycloalkyl. In some embodiments, optionally substituted heterocyclyldisclosed herein are optionally substituted 3-7 membered heterocyclyl,more preferably 5-6 membered heterocyclyl.

In some embodiments of the compounds of Formula (II), any of R¹ throughR⁸ may be selected from an alkyl substituted with one or moresubstituents selected from carboxyl (—CO₂H) or carboxylate (CO₂ ⁻),sulfo (SO₃H) or sulfonate (SO₃ ⁻) groups. In some such embodiments, thesubstituted alkyl is a substituted C₁, C₂, C₃, C₄, C₅, or C₆ alkyl. Insome such embodiments, the compounds of Formula (II) are alsorepresented by its salt form Formula (II′) with an organic or inorganiccation:

wherein K is an organic or inorganic cation, and n is an integerselected from 1 to 20.

In some embodiments of the compounds of Formula (II) or (II′), R^(Het)is a 9 membered heteroaryl optionally substituted with one or more R¹⁰.In some such embodiments, R^(Het) is selected from optionallysubstituted benzothiazolyl or benzoxazolyl, for example,2-benzothiazolyl or 2-benzoxazolyl. In some other embodiments, R^(Het)is a 6 membered heteroaryl optionally substituted with one or more R¹⁰.In one such embodiment, R^(Het) is an substituted pyridyl, for example,4-pyridyl with the structure:

It is understood that when R¹⁰ is connected to a pyridyl group bearing apositive charge on the nitrogen atom, R¹⁰ may contain a negative chargeso that R^(Het) as a whole is charge neutral. Alternatively, when R¹⁰ isconnected to a pyridyl group bearing a positive charge on the nitrogenatom, the compound described herein may contain a counterion so that thecompound as a whole is charge neutral. In some such embodiments, R¹⁰ isa substituted alkyl, for example, substituted C₁, C₂, C₃, C₄, C₅, or C₆alkyl. In some such embodiment, R¹⁰ is substituted with carboxyl(—CO₂H), carboxylate (CO₂ ⁻), sulfo (SO₃H), or sulfonate (SO₃ ⁻).

In some embodiments of the compounds of Formula (II) or (II′), the bondrepresented by a solid and dashed line

in Formula (II) is a double bond and the compounds are also representedby Formula (II-1) or (II′-1):

In some embodiments of the compounds of Formula (II), (II′), (II-1) or(II′-1), R² is alkyl. In one embodiment, R² is methyl. In some otherembodiments, R² is H.

In some embodiments of the compounds of Formula (II), (II′), (II-1) or(II′-1), at least one of R⁴ and R⁵ is alkyl. In some such embodiments,each R⁴ and R⁵ is alkyl. In one embodiment, both R⁴ and R⁵ are methyl.In some alternative embodiments, at least one of R⁴ and R⁵ is H. In onesuch embodiment, both R⁴ and R⁵ are H.

In some embodiments of the compounds of Formula (II) or (II′), the bondrepresented by a solid and dashed line

is a single bond and the compounds are also presented by Formula (II-2)or (II′-2):

In some embodiments of the compounds of Formula (II), (II′), (II-2) or(II′-2), at least one of R² and R³ is alkyl. In some furtherembodiments, both R² and R³ are alkyl. In one embodiment, both R² and R³are methyl. In some other embodiments, at least one of R² and R³ is H.In one embodiment, both R² and R³ are H.

In some embodiments of the compounds of Formula (II), (II′), (II-2) or(II′-2), at least one of R⁴ and R⁵ is H. In one such embodiment, both R⁴and R⁵ are H. In some alternative embodiments, at least one of R⁴ and R⁵is alkyl. In some such embodiments, each R⁴ and R⁵ is alkyl. In oneembodiment, both R⁴ and R⁵ are methyl.

In some embodiments of the compounds of Formula (II), (II′), (II-1),(II′-1), (II-2) or (II′-2), R⁶ is a substituted alkyl, for example,substituted C₁, C₂, C₃, C₄, C₅, or C₆ alkyl. In one embodiment, R⁶ isalkyl substituted with carboxyl. In some embodiments, R⁶ is an alkylsubstituted with —C(O)OR¹², and wherein R¹² is selected from the groupconsisting of optionally substituted alkyl, optionally substituted aryl,optionally substituted heteroaryl, and optionally substituted 3 to 7membered cycloalkyl. In one such embodiment, R¹² is an alkyl, forexample, methyl, ethyl, or t-, butyl. In some further embodiments, R⁶ isan alkyl substituted with —C(O)NR¹³R¹⁴ and wherein each R¹³ and R¹⁴ isindependently selected from H, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted heteroaryl and optionallysubstituted 3 to 7 membered cycloalkyl. In some further embodiments, R¹³and R¹⁴ is independently selected from an alkyl substituted with one ormore substituents selected from the group consisting of carboxyl,carboxylate, —C(O)OR¹¹, sulfo and sulfonate.

In some embodiments of the compounds of Formula (II), (II′), (II-1),(II′-1), (II-2) or (II′-2), R⁷ is H.

In some alternative embodiments of the compounds of Formula (II), (II′),(II-1), (II′-1), (II-2) or (II′-2), R⁶ and R⁷ are joined together withthe atoms to which they are attached to form an optionally substituted 3to 10 membered heterocyclyl, for example, an optionally substituted 6membered heterocyclyl. In some such embodiments, the optionallysubstituted heterocyclyl contains one heteroatom. In some suchembodiments, the optionally substituted heterocyclyl is substituted withone or more alkyl, for example, methyl.

In some embodiments of the compounds of Formula (II), (II′), (II-1),(II′-1), (II-2) or (II′-2), R¹ is H.

In some embodiments of the compounds of Formula (II), (II′), (II-1),(II′-1), (II-2) or (II′-2), R⁸ is H.

As understood by one of ordinary skill in the art, when a compound ofFormula (I) or (II) contains positively or negatively chargedsubstituent groups, it may also contains a negatively or positivelycharged counterion such that the compound as a whole is neutral.

Definition

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. The use of the term “including” as well as other forms, suchas “include”, “includes,” and “included,” is not limiting. The use ofthe term “having” as well as other forms, such as “have”, “has,” and“had,” is not limiting. As used in this specification, whether in atransitional phrase or in the body of the claim, the terms “comprise(s)”and “comprising” are to be interpreted as having an open-ended meaning.That is, the above terms are to be interpreted synonymously with thephrases “having at least” or “including at least.” For example, whenused in the context of a process, the term “comprising” means that theprocess includes at least the recited steps, but may include additionalsteps. When used in the context of a compound, composition, or device,the term “comprising” means that the compound, composition, or deviceincludes at least the recited features or components, but may alsoinclude additional features or components.

As used herein, common organic abbreviations are defined as follows:

-   -   Ac Acetyl    -   Ac₂O Acetic anhydride    -   aq. Aqueous    -   BOC or Boc tert-Butoxycarbonyl    -   BOP (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium        hexafluorophosphate    -   cat. Catalytic    -   ° C. Temperature in degrees Centigrade    -   dATP Deoxyadenosine triphosphate    -   dCTP Deoxycytidine triphosphate    -   dGTP Deoxyguanosine triphosphate    -   dTTP Deoxythymidine triphosphate    -   ddNTP(s) Dideoxynucleotide(s)    -   DCM Methylene chloride    -   DMA Dimethylacetamide    -   DMF Dimethylformamide    -   Et Ethyl    -   EtOAc Ethyl acetate    -   ffC Fully functionalized Nucleotide Conjugate    -   g Gram(s)    -   h or hr Hour(s)    -   IPA Isopropyl Alcohol    -   LCMS Liquid chromatography-mass spectrometry    -   MeCN Acetonitrile    -   mL Milliliter(s)    -   PG Protecting group    -   Ph Phenyl    -   ppt Precipitate    -   PyBOP (Benzotriazol-1-yloxy)tripyrrolidinophosphonium        hexafluorophosphate    -   RT, rt Room temperature    -   SBS Sequencing by Synthesis    -   TEA Triethylamine    -   TEAB Tetraethylammonium bromide    -   TFA Trifluoroacetic acid    -   TRIS Tris(hydroxymethyl)aminomethane    -   Tert, t tertiary    -   THF Tetrahydrofuran    -   TLC Thin Layer Chromatography    -   TSTU O—(N-Succinimidyl)-N,N,N′,N′-tetramethyluronium        tetrafluoroborate    -   μL Microliter(s)

As used herein, the term “covalently attached” or “covalently bonded”refers to the forming of a chemical bonding that is characterized by thesharing of pairs of electrons between atoms. For example, a covalentlyattached polymer coating refers to a polymer coating that forms chemicalbonds with a functionalized surface of a substrate, as compared toattachment to the surface via other means, for example, adhesion orelectrostatic interaction. It will be appreciated that polymers that areattached covalently to a surface can also be bonded via means inaddition to covalent attachment.

The term “halogen” or “halo,” as used herein, means any one of theradio-stable atoms of column 7 of the Periodic Table of the Elements,e.g., fluorine, chlorine, bromine, or iodine, with fluorine and chlorinebeing preferred.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that is fully saturated (i.e., contains no double or triplebonds). The alkyl group may have 1 to 20 carbon atoms (whenever itappears herein, a numerical range such as “1 to 20” refers to eachinteger in the given range; e.g., “1 to 20 carbon atoms” means that thealkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbonatoms, etc., up to and including 20 carbon atoms, although the presentdefinition also covers the occurrence of the term “alkyl” where nonumerical range is designated). The alkyl group may also be a mediumsize alkyl having 1 to 9 carbon atoms. The alkyl group could also be alower alkyl having 1 to 6 carbon atoms. The alkyl group may bedesignated as “C₁₋₄ alkyl” or similar designations. By way of exampleonly, “C₁₋₆ alkyl” indicates that there are one to six carbon atoms inthe alkyl chain, i.e., the alkyl chain is selected from the groupconsisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, and t-butyl. Typical alkyl groups include, but are in no waylimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiarybutyl, pentyl, hexyl, and the like.

As used herein, “alkoxy” refers to the formula —OR wherein R is an alkylas is defined above, such as “C₁₋₉ alkoxy”, including but not limited tomethoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy,iso-butoxy, sec-butoxy, and tert-butoxy, and the like.

As used herein, “alkenyl” refers to a straight or branched hydrocarbonchain containing one or more double bonds. The alkenyl group may have 2to 20 carbon atoms, although the present definition also covers theoccurrence of the term “alkenyl” where no numerical range is designated.The alkenyl group may also be a medium size alkenyl having 2 to 9 carbonatoms. The alkenyl group could also be a lower alkenyl having 2 to 6carbon atoms. The alkenyl group may be designated as “C₂₋₆ alkenyl” orsimilar designations. By way of example only, “C₂₋₆ alkenyl” indicatesthat there are two to six carbon atoms in the alkenyl chain, i.e., thealkenyl chain is selected from the group consisting of ethenyl,propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl,buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl,1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl,buta-1,2,-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groupsinclude, but are in no way limited to, ethenyl, propenyl, butenyl,pentenyl, and hexenyl, and the like.

As used herein, “alkynyl” refers to a straight or branched hydrocarbonchain containing one or more triple bonds. The alkynyl group may have 2to 20 carbon atoms, although the present definition also covers theoccurrence of the term “alkynyl” where no numerical range is designated.The alkynyl group may also be a medium size alkynyl having 2 to 9 carbonatoms. The alkynyl group could also be a lower alkynyl having 2 to 6carbon atoms. The alkynyl group may be designated as “C₂₋₆ alkynyl” orsimilar designations. By way of example only, “C₂₋₆ alkynyl” indicatesthat there are two to six carbon atoms in the alkynyl chain, i.e., thealkynyl chain is selected from the group consisting of ethynyl,propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and2-butynyl. Typical alkynyl groups include, but are in no way limited to,ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and the like.

As used herein, “heteroalkyl” refers to a straight or branchedhydrocarbon chain containing one or more heteroatoms, that is, anelement other than carbon, including but not limited to, nitrogen,oxygen and sulfur, in the chain backbone. The heteroalkyl group may have1 to 20 carbon atom, although the present definition also covers theoccurrence of the term “heteroalkyl” where no numerical range isdesignated. The heteroalkyl group may also be a medium size heteroalkylhaving 1 to 9 carbon atoms. The heteroalkyl group could also be a lowerheteroalkyl having 1 to 6 carbon atoms. The heteroalkyl group may bedesignated as “C₁₋₆ heteroalkyl” or similar designations. Theheteroalkyl group may contain one or more heteroatoms. By way of exampleonly, “C₁₋₆ heteroalkyl” indicates that there are one to six carbonatoms in the heteroalkyl chain and additionally one or more heteroatomsin the backbone of the chain.

The term “aromatic” refers to a ring or ring system having a conjugatedpi electron system and includes both carbocyclic aromatic (e.g., phenyl)and heterocyclic aromatic groups (e.g., pyridine). The term includesmonocyclic or fused-ring polycyclic (i.e., rings which share adjacentpairs of atoms) groups provided that the entire ring system is aromatic.

As used herein, “aryl” refers to an aromatic ring or ring system (i.e.,two or more fused rings that share two adjacent carbon atoms) containingonly carbon in the ring backbone. When the aryl is a ring system, everyring in the system is aromatic. The aryl group may have 6 to 18 carbonatoms, although the present definition also covers the occurrence of theterm “aryl” where no numerical range is designated. In some embodiments,the aryl group has 6 to 10 carbon atoms. The aryl group may bedesignated as “C₆₋₁₀ aryl,” “C₆ or C₁₀ aryl,” or similar designations.Examples of aryl groups include, but are not limited to, phenyl,naphthyl, azulenyl, and anthracenyl.

An “aralkyl” or “arylalkyl” is an aryl group connected, as asubstituent, via an alkylene group, such as “C₇₋₁₄ aralkyl” and thelike, including but not limited to benzyl, 2-phenylethyl,3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group isa lower alkylene group (i.e., a C₁₋₆ alkylene group).

As used herein, “heteroaryl” refers to an aromatic ring or ring system(i.e., two or more fused rings that share two adjacent atoms) thatcontain(s) one or more heteroatoms, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen and sulfur, inthe ring backbone. When the heteroaryl is a ring system, every ring inthe system is aromatic. The heteroaryl group may have 5-18 ring members(i.e., the number of atoms making up the ring backbone, including carbonatoms and heteroatoms), although the present definition also covers theoccurrence of the term “heteroaryl” where no numerical range isdesignated. In some embodiments, the heteroaryl group has 5 to 10 ringmembers or 5 to 7 ring members. The heteroaryl group may be designatedas “5-7 membered heteroaryl,” “5-10 membered heteroaryl,” or similardesignations. Examples of heteroaryl rings include, but are not limitedto, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl,imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl,thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,quinolinyl, isoquinlinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl,indolyl, isoindolyl, and benzothienyl.

A “heteroaralkyl” or “heteroarylalkyl” is heteroaryl group connected, asa substituent, via an alkylene group. Examples include but are notlimited to 2-thienylmethyl, 3-thienylmethyl, furylmethyl, thienylethyl,pyrrolylalkyl, pyridylalkyl, isoxazollylalkyl, and imidazolylalkyl. Insome cases, the alkylene group is a lower alkylene group (i.e., a C₁₋₆alkylene group).

As used herein, “carbocyclyl” means a non-aromatic cyclic ring or ringsystem containing only carbon atoms in the ring system backbone. Whenthe carbocyclyl is a ring system, two or more rings may be joinedtogether in a fused, bridged or spiro-connected fashion. Carbocyclylsmay have any degree of saturation provided that at least one ring in aring system is not aromatic. Thus, carbocyclyls include cycloalkyls,cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have 3 to 20carbon atoms, although the present definition also covers the occurrenceof the term “carbocyclyl” where no numerical range is designated. Thecarbocyclyl group may also be a medium size carbocyclyl having 3 to 10carbon atoms. The carbocyclyl group could also be a carbocyclyl having 3to 6 carbon atoms. The carbocyclyl group may be designated as “C₃₋₆carbocyclyl” or similar designations. Examples of carbocyclyl ringsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicycle[2.2.2]octanyl,adamantyl, and spiro[4.4]nonanyl.

As used herein, “cycloalkyl” means a fully saturated carbocyclyl ring orring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

As used herein, “heterocyclyl” means a non-aromatic cyclic ring or ringsystem containing at least one heteroatom in the ring backbone.Heterocyclyls may be joined together in a fused, bridged orspiro-connected fashion. Heterocyclyls may have any degree of saturationprovided that at least one ring in the ring system is not aromatic. Theheteroatom(s) may be present in either a non-aromatic or aromatic ringin the ring system. The heterocyclyl group may have 3 to 20 ring members(i.e., the number of atoms making up the ring backbone, including carbonatoms and heteroatoms), although the present definition also covers theoccurrence of the term “heterocyclyl” where no numerical range isdesignated. The heterocyclyl group may also be a medium sizeheterocyclyl having 3 to 10 ring members. The heterocyclyl group couldalso be a heterocyclyl having 3 to 6 ring members. The heterocyclylgroup may be designated as “3-6 membered heterocyclyl” or similardesignations. In preferred six membered monocyclic heterocyclyls, theheteroatom(s) are selected from one up to three of O, N or S, and inpreferred five membered monocyclic heterocyclyls, the heteroatom(s) areselected from one or two heteroatoms selected from O, N, or S. Examplesof heterocyclyl rings include, but are not limited to, azepinyl,acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl,imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl,piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl,pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl,1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl,1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl,hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl,1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl,oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl,isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl,thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, andtetrahydroquinoline.

An “O-carboxy” group refers to a “—OC(═O)R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, asdefined herein.

A “C-carboxy” group refers to a “—C(═O)OR” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, asdefined herein. A non-limiting example includes carboxyl (i.e.,—C(═O)OH).

A “cyano” group refers to a “—CN” group.

A “sulfonyl” group refers to an “—SO₂R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, asdefined herein.

A “sulfo” or “sulfonyl hydroxide” group refers to a “—S(═O)₂—OH” group.

A “sulfino” group refers to a “—S(═O)OH” group.

A “sulfonate” group refers to —SO₃ ⁻.

An “S-sulfonamido” group refers to a “—SO₂NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

An “N-sulfonamido” group refers to a “—N(R_(A))SO₂R_(B)” group in whichR_(A) and R_(b) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

A “C-amido” group refers to a “—C(═O)NR_(A)R_(B)” group in which R_(A)and R_(B) are each independently selected from hydrogen, C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 3-10 membered heterocyclyl, as defined herein.

An “N-amido” group refers to a “—N(R_(A))C(═O)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

An “amino” group refers to a “—NR_(A)R_(B)” group in which R_(A) andR_(B) are each independently selected from hydrogen, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 3-10 membered heterocyclyl, as defined herein. Anon-limiting example includes free amino (i.e., —NH₂).

An “aminoalkyl” group refers to an amino group connected via an alkylenegroup.

An “alkoxyalkyl” group refers to an alkoxy group connected via analkylene group, such as a “C₂₋₈ alkoxyalkyl” and the like.

As used herein, a substituted group is derived from the unsubstitutedparent group in which there has been an exchange of one or more hydrogenatoms for another atom or group. Unless otherwise indicated, when agroup is deemed to be “substituted,” it is meant that the group issubstituted with one or more substituents independently selected fromC₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl, C₁-C₆ heteroalkyl, C₃-C₇carbocyclyl (optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),C₃-C₇-carbocyclyl-C₁-C₆-alkyl (optionally substituted with halo, C₁-C₆alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 3-10membered heterocyclyl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 3-10 memberedheterocyclyl-C₁-C₆-alkyl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), aryl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), aryl(C₁-C₆)alkyl (optionally substituted with halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10membered heteroaryl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10 memberedheteroaryl(C₁-C₆)alkyl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), halo, cyano,hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkoxy(C₁-C₆)alkyl (i.e., ether), aryloxy,sulfhydryl (mercapto), halo(C₁-C₆)alkyl (e.g., —CF₃), halo(C₁-C₆)alkoxy(e.g., —OCF₃), C₁-C₆ alkylthio, arylthio, amino, amino(C₁-C₆)alkyl,nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl,cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl,sulfo, sulfino, sulfonate, and oxo (═O). Wherever a group is describedas “optionally substituted” that group can be substituted with the abovesubstituents.

As understood by one of ordinary skill in the art, if a compoundcontains positively or negatively charged substituent groups, forexample, SO₃ ⁻, it may also contains a negatively or positively chargedcounterion such that the compound as a whole is neutral.

It is to be understood that certain radical naming conventions caninclude either a mono-radical or a di-radical, depending on the context.For example, where a substituent requires two points of attachment tothe rest of the molecule, it is understood that the substituent is adi-radical. For example, a substituent identified as alkyl that requirestwo points of attachment includes di-radicals such as —CH₂—, —CH₂CH₂—,—CH₂CH(CH₃)CH₂—, and the like. Other radical naming conventions clearlyindicate that the radical is a di-radical such as “alkylene” or“alkenylene.”

When two “adjacent” R groups are said to form a ring “together with theatom to which they are attached,” it is meant that the collective unitof the atoms, intervening bonds, and the two R groups are the recitedring. For example, when the following substructure is present:

and R¹ and R² are defined as selected from the group consisting ofhydrogen and alkyl, or R¹ and R² together with the atoms to which theyare attached form an aryl or carbocyclyl, it is meant that R¹ and R² canbe selected from hydrogen or alkyl, or alternatively, the substructurehas structure:

where A is an aryl ring or a carbocyclyl containing the depicted doublebond.

Labeled Nucleotides

The dye compounds described herein are suitable for attachment tosubstrate moieties. Substrate moieties can be virtually any molecule orsubstance to which the fluorescent dyes described herein can beconjugated and, by way of non-limiting example, may include nucleosides,nucleotides, polynucleotides, carbohydrates, ligands, particles, solidsurfaces, organic and inorganic polymers and combinations or assemblagesthereof, such as chromosomes, nuclei, living cells and the like. Thedyes can be conjugated by an optional linker by a variety of meansincluding hydrophobic attraction, ionic attraction and covalentattachment. Particularly the dyes are conjugated to the substrate bycovalent attachment. More particularly the covalent attachment is bymeans of a linker group. In some instances, such labeled nucleotides arealso referred to as “modified nucleotides.”

A particular useful application of the new fluorescent dyes with longStokes shift as described herein is for labeling biomolecules, forexample, nucleotides or oligonucleotides. Some embodiments of thepresent application are directed to a nucleotide or oligonucleotidelabeled with the new fluorescent compounds as described herein.

Fluorescent dye molecules with improved fluorescence properties (such asStokes shift, fluorescence intensity, position of fluorescence maximumand shape of fluorescence band) can improve the speed and accuracy ofnucleic acid sequencing. Stokes Shift is a key aspect in the detectionof the fluorescence in biological applications. For example, thedetection of emitted light can be difficult to distinguish from theexcitation light when using fluorophores with absorption andfluorescence max very close to each other (i.e., small Stokes shift),because the excitation and emission wavelengths greatly overlap. Incontrast, fluorophores with large Stokes shifts are easy to distinguishbecause of the greater separation between the excitation and emissionwavelengths. The Stokes shift is especially critical in multiplexfluorescence applications, because the emission wavelength of onefluorophore may overlap, and therefore excite, another fluorophore inthe same sample. In addition, fluorescence signal intensity isespecially important when measurements are made in water basedbiological buffers and/or at higher temperature as fluorescence of mostdyes is significantly lower at such conditions. Moreover, the nature ofthe base to which a dye is attached also affects the fluorescencemaximum, fluorescence intensity and other spectral dye properties. Thesequence specific interactions between the fluorescent dye and thenucleobase can be tailored by specific design of the fluorescent dyes.Optimization of the structure of the fluorescent dyes can improve theirfluorescent properties and also improve the efficiency of nucleotideincorporation, reduce the level of sequencing errors and decrease theusage of reagents in, and therefore the costs of, nucleic acidsequencing.

The attachment to the biomolecules may be via R⁶, R⁸, R^(10a), R^(10b),R^(10c) or R¹⁰ moiety of the compound of Formula (I) or Formula (II). Insome embodiments, R⁶, R⁸, R^(10a), R^(10b), R^(10c) or R¹⁰ is asubstituted alkyl, for example alkyl substituted with carboxyl (i.e.,—CO₂H) or an activated form of carboxyl group, for example, amide orester, which may be used for attachment to the amino group of thebiomolecules. In one embodiment, R⁶, R⁸, R^(10a), R^(10b), R^(10c) orR¹⁰ may contain an activated ester or amide residue most suitable forfurther amide/peptide bond formation. The term “activated ester” as usedherein, refers to a carboxyl group derivative which is capable ofreacting in mild conditions, for example, with a compound containing anamino group. Non-limiting examples of activated esters include but notlimited to p-nitrophenyl, pentafluorophenyl and succinimido esters.

In some embodiments, the dye compounds may be covalently attached tooligonucleotides or nucleotides via the nucleotide base. For example,the labeled nucleotide or oligonucleotide may have the label attached tothe C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base through a linker moiety. The labeled nucleotide oroligonucleotide may also have a 3′-OH blocking group covalently attachedto the ribose or deoxyribose sugar of the nucleotide.

Linkers

The dye compounds as disclosed herein may include a reactive linkergroup at one of the substituent positions for covalent attachment of thecompound to another molecule. Reactive linking groups are moietiescapable of forming a covalent bond. In a particular embodiment thelinker may be a cleavable linker. Use of the term “cleavable linker” isnot meant to imply that the whole linker is required to be removed. Thecleavage site can be located at a position on the linker that ensuresthat part of the linker remains attached to the dye and/or substratemoiety after cleavage. Cleavable linkers may be, by way of non-limitingexample, electrophilically cleavable linkers, nucleophilically cleavablelinkers, photocleavable linkers, cleavable under reductive conditions(for example disulfide or azide containing linkers), oxidativeconditions, cleavable via use of safety-catch linkers and cleavable byelimination mechanisms. The use of a cleavable linker to attach the dyecompound to a substrate moiety ensures that the label can, if required,be removed after detection, avoiding any interfering signal indownstream steps.

Non-limiting examples of linker groups include those disclosed in PCTPublication No. WO2004/018493 (herein incorporated by reference), whichconnect the bases of nucleotides to labels such as, for example, the newfluorescent compounds described herein. These linkers may be cleavedusing water-soluble phosphines or water-soluble transition metalcatalysts formed from a transition metal and at least partiallywater-soluble ligands. In aqueous solution the latter form at leastpartially water-soluble transition metal complexes. Additional suitablelinkers that may be used include those disclosed in PCT Publication No.WO2004/018493 and WO 2007/020457 (both of which are herein incorporatedby references. It was discovered that by altering, and in particularincreasing, the length of the linker between a fluorescent dye(fluorophore) and the guanine base, by introducing a polyethylene glycolspacer group, it is possible to increase the fluorescence intensitycompared to the same fluorophore attached to the guanine base throughother linkages known in the art. The design of the linkers, andespecially their increased length, also allows improvements in thebrightness of fluorophores attached to the guanine bases of guanosinenucleotides when incorporated into polynucleotides such as DNA. Thus,when the dye is for use in any method of analysis which requiresdetection of a fluorescent dye label attached to a guanine-containingnucleotide, it is advantageous if the linker comprises a spacer group offormula —((CH₂)₂O)_(n)—, wherein n is an integer between 2 and 50, asdescribed in WO 2007/020457.

Nucleosides and nucleotides may be labeled at sites on the sugar ornucleobase. As understood by one of ordinary skill in the art, a“nucleotide” consists of a nitrogenous base, a sugar, and one or morephosphate groups. In RNA the sugar is ribose and in DNA is adeoxyribose, i.e. a sugar lacking a hydroxyl group that is present inribose. The nitrogenous base is a derivative of purine or pyrimidine.The purines are adenine (A) and guanine (G), and the pyrimidines arecytosine (C) and thymine (T) or in the context of RNA, uracil (U). TheC-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of apurine. A nucleotide is also a phosphate ester of a nucleoside, withesterification occurring on the hydroxyl group attached to the C-3 orC-5 of the sugar. Nucleotides are usually mono, di- or triphosphates.

A “nucleoside” is structurally similar to a nucleotide but is missingthe phosphate moieties. An example of a nucleoside analog would be onein which the label is linked to the base and there is no phosphate groupattached to the sugar molecule.

Although the base is usually referred to as a purine or pyrimidine, theskilled person will appreciate that derivatives and analogues areavailable which do not alter the capability of the nucleotide ornucleoside to undergo Watson-Crick base pairing. “Derivative” or“analogue” means a compound or molecule whose core structure is the sameas, or closely resembles that of a parent compound but which has achemical or physical modification, such as, for example, a different oradditional side group, which allows the derivative nucleotide ornucleoside to be linked to another molecule. For example, the base maybe a deazapurine. The derivatives should be capable of undergoingWatson-Crick pairing. “Derivative” and “analogue” also mean a syntheticnucleotide or nucleoside derivative having modified base moieties and/ormodified sugar moieties. Such derivatives and analogues are discussedin, for example, Scheit, Nucleotide analogs (John Wiley & Son, 1980) andUhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotide analoguescan also comprise modified phosphodiester linkages includingphosphorothioate, phosphorodithioate, alkyl-phosphonate,phosphoranilidate, phosphoramidate linkages and the like.

The dye may be attached to any position on the nucleotide base, througha linker, provided that Watson-Crick base pairing can still be carriedout. Particular nucleobase labeling sites include the C5 position of apyrimidine base or the C7 position of a 7-deaza purine base. Asdescribed above a linker group may be used to covalently attach a dye tothe nucleoside or nucleotide.

In particular embodiments the labeled nucleoside or nucleotide may beenzymatically incorporable and enzymatically extendable. Accordingly alinker moiety may be of sufficient length to connect the nucleotide tothe compound such that the compound does not significantly interferewith the overall binding and recognition of the nucleotide by a nucleicacid replication enzyme. Thus, the linker can also comprise a spacerunit. The spacer distances, for example, the nucleotide base from acleavage site or label.

Nucleosides or nucleotides labeled with the new fluorescent dyesdescribed herein may have the formula:

where Dye is a dye compound, B is a nucleobase, such as, for exampleuracil, thymine, cytosine, adenine, guanine and the like and L is anoptional linker group which may or may not be present. R′ can be H,monophosphate, diphosphate, triphosphate, thiophosphate, a phosphateester analog, —O— attached to a reactive phosphorous containing group or—O— protected by a blocking group. R″ can be H, OH, a phosphoramidite ora 3′-OH blocking group and R′″ is H or OH; where R″ is phosphoramidite,R′ is an acid-cleavable hydroxyl protecting group which allowssubsequent monomer coupling under automated synthesis conditions.

In some instances, the blocking group is separate and independent of thedye compound, i.e. not attached to it. Alternatively, the dye maycomprise all or part of the 3′-OH blocking group. Thus R″ can be a 3′-OHblocking group which may or may not comprise the dye compound. Inadditional alternative embodiments, there is no blocking group on the 3′carbon of the pentose sugar and the dye (or dye and linker construct)attached to the base, for example, can be of a size or structuresufficient to act as a block to the incorporation of a furthernucleotide from a point other than the 3′ site. Thus the block can bedue to steric hindrance or can be due to a combination of size, chargeand structure.

The use of a blocking group allows polymerization to be controlled, suchas by stopping extension when a modified nucleotide is incorporated. Ifthe blocking effect is reversible, for example by way of non-limitingexample by changing chemical conditions or by removal of a chemicalblock, extension can be stopped at certain points and then allowed tocontinue. Non-limiting examples of 3′-OH blocking groups include thosedisclosed in WO 2004/018497 and WO2014/139596, which are herebyincorporated by references. For example the blocking group may beazidomethyl (—CH₂N₃) or substituted azidomethyl (e.g., —CH(CHF₂)N₃ orCH(CH₂F)N₃), or allyl.

In a particular embodiment the linker and blocking group are bothpresent and are separate moieties which are both cleavable undersubstantially similar conditions. Thus deprotection and deblockingprocesses may be more efficient since only a single treatment will berequired to remove both the dye compound and the blocking group.

The present disclosure also directs to encompassing polynucleotidesincorporating dye compounds described herein. Such polynucleotides maybe DNA or RNA comprised respectively of deoxyribonucleotides orribonucleotides joined in phosphodiester linkage. Polynucleotides maycomprise naturally occurring nucleotides, non-naturally occurring (ormodified) nucleotides other than the labeled nucleotides describedherein or any combination thereof, provided that at least one nucleotidelabeled with a dye compound, according to the present application ispresent. Polynucleotides may also include non-natural backbone linkagesand/or non-nucleotide chemical modifications. Chimeric structurescomprised of mixtures of ribonucleotides and deoxyribonucleotidescomprising at least one labeled nucleotide are also contemplated.

Non-limiting exemplary labeled nucleotides as described herein include:

wherein: L represents a linker and R represents a sugar residue asdescribed above.

In some embodiments, non-limiting exemplary fluorescent dye conjugatesare shown below:

Kits

Some embodiments disclosed herein are kits including nucleosides and/ornucleotides labeled with the new fluorescent dyes described herein. Suchkits will generally include at least one nucleotide or nucleosidelabeled with a dye together with at least one further component. Thefurther component(s) may be further modified or unmodified nucleotidesor nucleosides. For example, nucleotides labeled with dyes may besupplied in combination with unlabeled or native nucleotides, and/orwith fluorescently labeled nucleotides or any combination thereof.Combinations of nucleotides may be provided as separate individualcomponents or as nucleotide mixtures. In some embodiments, the kitscomprise one or more nucleotides wherein at least one nucleotide is anucleotide labeled with a new fluorescent compound described herein. Thekits may comprise two or more labeled nucleotides. The nucleotides maybe labeled with two or more fluorescent labels. Two or more of thelabels may be excited using a single excitation source, which may be alaser.

The kits may contain four nucleotides, where the first of fournucleotides is labeled with a compound as disclosed herein, and thesecond, third, and fourth nucleotides are each may be labeled with adifferent compound, wherein each compound has a distinct fluorescencemaximum and each of the compounds is distinguishable from the otherthree compounds. The kits may be such that two or more of the compoundshave a similar absorbance maximum but different Stokes shift.

The fluorescent dye compounds, labeled nucleotides or kits describedherein may be used in sequencing, expression analysis, hybridizationanalysis, genetic analysis, RNA analysis or protein binding assays. Theuse may be on an automated sequencing instrument. The sequencinginstrument may contain two lasers operating at different wavelengths.

Where kits comprise a plurality, particularly two, more particularlyfour, nucleotides labeled with a dye compound, the different nucleotidesmay be labeled with different dye compounds, or one may be dark, with nodye compounds. Where the different nucleotides are labeled withdifferent dye compounds it is a feature of the kits that said dyecompounds are spectrally distinguishable fluorescent dyes. As usedherein, the term “spectrally distinguishable fluorescent dyes” refers tofluorescent dyes that emit fluorescent energy at wavelengths that can bedistinguished by fluorescent detection equipment (for example, acommercial capillary based DNA sequencing platform) when two or moresuch dyes are present in one sample. When two nucleotides labeled withfluorescent dye compounds are supplied in kit form, the spectrallydistinguishable fluorescent dyes can be excited at the same wavelength,such as, for example by the same laser in some embodiments. When fournucleotides labeled with fluorescent dye compounds are supplied in kitform, two of the spectrally distinguishable fluorescent dyes can both beexcited at one wavelength and the other two spectrally distinguishabledyes can both be excited at another wavelength in some embodiments.Particular excitation wavelengths are about 460 nm.

In some embodiments, at least one nucleotide may be labelled with a dyewhich excitable with two lasers with different wavelength.

In one embodiment a kit comprises a nucleotide labeled with a compounddescribed herein and a second nucleotide labeled with a second dyewherein the dyes have a difference in absorbance maximum of at least 10nm, particularly 20 nm to 50 nm. More particularly the two dye compoundshave Stokes shifts of between 15-40 nm or between 20-40 nm. As usedherein, the term “Stokes shift” is the distance between the peakabsorption and peak emission wavelengths.

In a further embodiment said kit further comprises two other nucleotideslabeled with fluorescent dyes wherein said dyes are excited by thelasers at about 440 nm to about 560 nm.

In an alternative embodiment, the kits may contain nucleotides where thesame base is labeled with two different compounds. A first nucleotidemay be labeled with a compound described herein. A second nucleotide maybe labeled with a spectrally distinct compound, for example a ‘red’ dyeabsorbing at greater than 600 nm. A third nucleotide may be labeled as amixture of the fluorescent dye compound described herein and thespectrally distinct compound, and the fourth nucleotide may be ‘dark’and contain no label. In simple terms therefore the nucleotides 1-4 maybe labeled ‘green’, ‘red’, ‘red/green’, and dark. To simplify theinstrumentation further, four nucleotides can be labeled with a two dyesexcited with a single laser, and thus the labeling of nucleotides 1-4may be ‘green 1’, ‘green 2’ ‘green 1/green 2’, and dark.

In other embodiments the kits may include a polymerase enzyme capable ofcatalyzing incorporation of the nucleotides into a polynucleotide. Othercomponents to be included in such kits may include buffers and the like.The nucleotides labeled with the new fluorescent dyes described herein,and other any nucleotide components including mixtures of differentnucleotides, may be provided in the kit in a concentrated form to bediluted prior to use. In such embodiments a suitable dilution buffer mayalso be included.

Methods of Sequencing

Nucleotides (or nucleosides) comprising a new fluorescent dye describedherein may be used in any method of analysis which requires detection ofa fluorescent label attached to a nucleotide or nucleoside, whether onits own or incorporated into or associated with a larger molecularstructure or conjugate. Some embodiments of the present application aredirected to methods of sequencing including: (a) incorporating at leastone labeled nucleotide as described herein into a polynucleotide; and(b) detecting the labeled nucleotide(s) incorporated into thepolynucleotide by detecting the fluorescent signal from the newfluorescent dye attached to said modified nucleotide(s).

In some embodiments, at least one labeled nucleotide is incorporatedinto a polynucleotide in the synthetic step by the action of apolymerase enzyme. However, other methods of incorporating labelednucleotides to polynucleotides, such as chemical oligonucleotidesynthesis or ligation of labeled oligonucleotides to unlabeledoligonucleotides, are not excluded. Therefore, the term “incorporating”a nucleotide into a polynucleotide encompasses polynucleotide synthesisby chemical methods as well as enzymatic methods.

In all embodiments of the methods, the detection step may be carried outwhilst the polynucleotide strand into which the labeled nucleotides areincorporated is annealed to a template strand, or after a denaturationstep in which the two strands are separated. Further steps, for examplechemical or enzymatic reaction steps or purification steps, may beincluded between the synthetic step and the detection step. Inparticular, the target strand incorporating the labeled nucleotide(s)may be isolated or purified and then processed further or used in asubsequent analysis. By way of example, target polynucleotides labeledwith modified nucleotide(s) as described herein in a synthetic step maybe subsequently used as labeled probes or primers. In other embodimentsthe product of the synthetic step (a) may be subject to further reactionsteps and, if desired, the product of these subsequent steps purified orisolated.

Suitable conditions for the synthetic step will be well known to thosefamiliar with standard molecular biology techniques. In one embodimentthe synthetic step may be analogous to a standard primer extensionreaction using nucleotide precursors, including modified nucleotidesaccording to the present disclosure, to form an extended target strandcomplementary to the template strand in the presence of a suitablepolymerase enzyme. In other embodiments the synthetic step may itselfform part of an amplification reaction producing a labeled doublestranded amplification product comprised of annealed complementarystrands derived from copying of the target and template polynucleotidestrands. Other exemplary “synthetic” steps include nick translation,strand displacement polymerization, random primed DNA labeling etc. Thepolymerase enzyme used in the synthetic step must be capable ofcatalyzing the incorporation of modified nucleotides according to thepresent disclosure. Otherwise, the precise nature of the polymerase isnot particularly limited but may depend upon the conditions of thesynthetic reaction. By way of example, if the synthetic reaction iscarried out using thermocycling then a thermostable polymerase isrequired, whereas this may not be essential for standard primerextension reactions. Suitable thermostable polymerases which are capableof incorporating the modified nucleotides according to the presentdisclosure include those described in WO 2005/024010 or WO 2006/120433.In synthetic reactions which are carried out at lower temperatures suchas 37° C., polymerase enzymes need not necessarily be thermostablepolymerases, therefore the choice of polymerase will depend on a numberof factors such as reaction temperature, pH, strand-displacing activityand the like.

In specific non-limiting embodiments, the modified nucleotides ornucleosides labeled with the new fluorescent dyes with longer Stokesshift according to the present application may be used in a method ofnucleic acid sequencing, re-sequencing, whole genome sequencing, singlenucleotide polymorphism scoring, any other application involving thedetection of the modified nucleotide or nucleoside when incorporatedinto a polynucleotide, or any other application requiring the use ofpolynucleotides labeled with the modified nucleotides comprisingfluorescent dyes according to the present application.

In a particular embodiment the present application provides use ofmodified nucleotides comprising dye compounds described herein in apolynucleotide “sequencing-by-synthesis” reaction.Sequencing-by-synthesis generally involves sequential addition of one ormore nucleotides or oligonucleotides to a growing polynucleotide chainin the 5′ to 3′ direction using a polymerase or ligase in order to forman extended polynucleotide chain complementary to the template nucleicacid to be sequenced. The identity of the base present in one or more ofthe added nucleotide(s) is determined in a detection or “imaging” step.The identity of the added base may be determined after each nucleotideincorporation step. The sequence of the template may then be inferredusing conventional Watson-Crick base-pairing rules. The use of themodified nucleotides labeled with dyes according to the presentdisclosure for determination of the identity of a single base may beuseful, for example, in the scoring of single nucleotide polymorphisms,and such single base extension reactions are within the scope of thisapplication.

In an embodiment, the sequence of a template polynucleotide isdetermined by detecting the incorporation of one or more nucleotidesinto a nascent strand complementary to the template polynucleotide to besequenced through the detection of fluorescent label(s) attached to theincorporated nucleotide(s). Sequencing of the template polynucleotide isprimed with a suitable primer (or prepared as a hairpin construct whichwill contain the primer as part of the hairpin), and the nascent chainis extended in a stepwise manner by addition of nucleotides to the 3′end of the primer in a polymerase-catalyzed reaction.

In particular embodiments each of the different nucleotide triphosphates(A, T, G and C) may be labeled with a unique fluorophore and alsocomprises a blocking group at the 3′ position to prevent uncontrolledpolymerization. Alternatively, one of the four nucleotides may beunlabeled (dark). The polymerase enzyme incorporates a nucleotide intothe nascent chain complementary to the template polynucleotide, and theblocking group prevents further incorporation of nucleotides. Anyunincorporated nucleotides are removed and the fluorescent signal fromeach incorporated nucleotide is “read” optically by suitable means, suchas a charge-coupled device using laser excitation and suitable emissionfilters. The 3′-blocking group and fluorescent dye compounds are thenremoved (deprotected), particularly by the same chemical or enzymaticmethod, to expose the nascent chain for further nucleotideincorporation. Typically, the identity of the incorporated nucleotidewill be determined after each incorporation step but this is notstrictly essential. Similarly, U.S. Pat. No. 5,302,509 discloses amethod to sequence polynucleotides immobilized on a solid support. Themethod relies on the incorporation of fluorescently labeled, 3′-blockednucleotides A, G, C and T into a growing strand complementary to theimmobilized polynucleotide, in the presence of DNA polymerase. Thepolymerase incorporates a base complementary to the targetpolynucleotide, but is prevented from further addition by the3′-blocking group. The label of the incorporated nucleotide can then bedetermined and the blocking group removed by chemical cleavage to allowfurther polymerization to occur. The nucleic acid template to besequenced in a sequencing-by-synthesis reaction may be anypolynucleotide that it is desired to sequence. The nucleic acid templatefor a sequencing reaction will typically comprise a double strandedregion having a free 3′ hydroxyl group which serves as a primer orinitiation point for the addition of further nucleotides in thesequencing reaction. The region of the template to be sequenced willoverhang this free 3′ hydroxyl group on the complementary strand. Theoverhanging region of the template to be sequenced may be singlestranded but can be double-stranded, provided that a “nick is present”on the strand complementary to the template strand to be sequenced toprovide a free 3′ OH group for initiation of the sequencing reaction. Insuch embodiments sequencing may proceed by strand displacement. Incertain embodiments a primer bearing the free 3′ hydroxyl group may beadded as a separate component (e.g. a short oligonucleotide) whichhybridizes to a single-stranded region of the template to be sequenced.Alternatively, the primer and the template strand to be sequenced mayeach form part of a partially self-complementary nucleic acid strandcapable of forming an intra-molecular duplex, such as for example ahairpin loop structure. Hairpin polynucleotides and methods by whichthey may be attached to solid supports are disclosed in PCT PublicationNos. WO 2001/057248 and WO 2005/047301. Nucleotides are addedsuccessively to the free 3′-hydroxyl group, resulting in synthesis of apolynucleotide chain in the 5′ to 3′ direction. The nature of the basewhich has been added may be determined, particularly but not necessarilyafter each nucleotide addition, thus providing sequence information forthe nucleic acid template. The term “incorporation” of a nucleotide intoa nucleic acid strand (or polynucleotide) in this context refers tojoining of the nucleotide to the free 3′ hydroxyl group of the nucleicacid strand via formation of a phosphodiester linkage with the 5′phosphate group of the nucleotide.

The nucleic acid template to be sequenced may be DNA or RNA, or even ahybrid molecule comprised of deoxynucleotides and ribonucleotides. Thenucleic acid template may comprise naturally occurring and/ornon-naturally occurring nucleotides and natural or non-natural backbonelinkages, provided that these do not prevent copying of the template inthe sequencing reaction.

In certain embodiments the nucleic acid template to be sequenced may beattached to a solid support via any suitable linkage method known in theart, for example via covalent attachment. In certain embodimentstemplate polynucleotides may be attached directly to a solid support(e.g. a silica-based support). However, in other embodiments the surfaceof the solid support may be modified in some way so as to allow eitherdirect covalent attachment of template polynucleotides, or to immobilizethe template polynucleotides through a hydrogel or polyelectrolytemultilayer, which may itself be non-covalently attached to the solidsupport.

Arrays in which polynucleotides have been directly attached tosilica-based supports are those for example disclosed in PCT PublicationNo. WO 2000/006770, wherein polynucleotides are immobilized on a glasssupport by reaction between a pendant epoxide group on the glass with aninternal amino group on the polynucleotide. In addition, PCT PublicationNo. WO2005/047301 discloses arrays of polynucleotides attached to asolid support, e.g. for use in the preparation of SMAs, by reaction of asulfur-based nucleophile with the solid support. A still further exampleof solid-supported template polynucleotides is where the templatepolynucleotides are attached to hydrogel supported upon silica-based orother solid supports. Silica-based supports are typically used tosupport hydrogels and hydrogel arrays as described in PCT PublicationNos. WO 00/31148, WO 01/01143, WO02/12566, WO 03/014392, WO 00/53812 andU.S. Pat. No. 6,465,178.

A particular surface to which template polynucleotides may beimmobilized is a polyacrylamide hydrogel. Polyacrylamide hydrogels aredescribed in the prior art, some of which is discussed above. Specifichydrogels that may be used in the present application include thosedescribed in WO 2005/065814 and U.S. Pub. No. 2014/0079923. In oneembodiment, the hydrogel is PAZAM (poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide)).

DNA template molecules can be attached to beads or microparticles forthe purposes of sequencing; for example as described in U.S. Pat. No.6,172,218. Further examples of the preparation of bead libraries whereeach bead contains different DNA sequences can be found in Margulies etal., Nature 437, 376-380 (2005); Shendure et al., Science.309(5741):1728-1732 (2005). Sequencing of arrays of such beads usingnucleotides as described is within the scope of the present application.

The template(s) to be sequenced may form part of an “array” on a solidsupport, in which case the array may take any convenient form. Thus, themethod of the present disclosure is applicable to all types of “highdensity” arrays, including single-molecule arrays, clustered arrays andbead arrays. Modified nucleotides labeled with dye compounds of thepresent application may be used for sequencing templates on essentiallyany type of array formed by immobilization of nucleic acid molecules ona solid support, and more particularly any type of high-density array.However, the modified nucleotides labeled with the new fluorescent dyesdescribed herein are particularly advantageous in the context ofsequencing of clustered arrays.

In multi-polynucleotide or clustered arrays, distinct regions on thearray comprise multiple polynucleotide template molecules. The term“clustered array” refers to an array wherein distinct regions or siteson the array comprise multiple polynucleotide molecules that are notindividually resolvable by optical means. Depending on how the array isformed each site on the array may comprise multiple copies of oneindividual polynucleotide molecule or even multiple copies of a smallnumber of different polynucleotide molecules (e.g. multiple copies oftwo complementary nucleic acid strands). Multi-polynucleotide orclustered arrays of nucleic acid molecules may be produced usingtechniques generally known in the art. By way of example, WO 98/44151and WO 00/18957 both describe methods of amplification of nucleic acidswherein both the template and amplification products remain immobilizedon a solid support in order to form arrays comprised of clusters or“colonies” of immobilized nucleic acid molecules. The nucleic acidmolecules present on the clustered arrays prepared according to thesemethods are suitable templates for sequencing using the modifiednucleotides labeled with the new fluorescent dyes described herein.

The modified nucleotides labeled with dye compounds of the presentapplication are also useful in sequencing of templates on singlemolecule arrays. The term “single molecule array” or “SMA” as usedherein refers to a population of polynucleotide molecules, distributed(or arrayed) over a solid support, wherein the spacing of any individualpolynucleotide from all others of the population is such that it ispossible to effect individual resolution of the polynucleotides. Thetarget nucleic acid molecules immobilized onto the surface of the solidsupport should thus be capable of being resolved by optical means. Thismeans that, within the resolvable area of the particular imaging deviceused, there must be one or more distinct signals, each representing onepolynucleotide.

This may be achieved wherein the spacing between adjacent polynucleotidemolecules on the array is at least 100 nm, more particularly at least250 nm, still more particularly at least 300 nm, even more particularlyat least 350 nm. Thus, each molecule is individually resolvable anddetectable as a single molecule fluorescent point, and fluorescence fromsaid single molecule fluorescent point also exhibits single stepphoto-bleaching.

The terms “individually resolved” and “individual resolution” are usedherein to specify that, when visualized, it is possible to distinguishone molecule on the array from its neighboring molecules. Separationbetween individual molecules on the array will be determined, in part,by the particular technique used to resolve the individual molecules.The general features of single molecule arrays will be understood byreference to PCT Publication Nos. WO 2000/006770 and WO 2001/057248.Although one application of the modified nucleotides of the presentdisclosure is in sequencing-by-synthesis reactions, the utility of suchlabeled nucleotides is not limited to such methods. In fact, thenucleotides may be used advantageously in any sequencing methodologywhich requires detection of fluorescent labels attached to nucleotidesincorporated into a polynucleotide.

In particular, the modified nucleotides labeled with dye compounds ofthe present application may be used in automated fluorescent sequencingprotocols, particularly fluorescent dye-terminator cycle sequencingbased on the chain termination sequencing method of Sanger andco-workers. Such methods generally use enzymes and cycle sequencing toincorporate fluorescently labeled dideoxynucleotides in a primerextension sequencing reaction. So called Sanger sequencing methods, andrelated protocols (Sanger-type), rely upon randomized chain terminationwith labeled dideoxynucleotides.

Thus, the present disclosure also encompasses modified nucleotideslabeled with dye compounds as described herein which aredideoxynucleotides lacking hydroxyl groups at both of the 3′ and 2′positions, such modified dideoxynucleotides being suitable for use inSanger type sequencing methods and the like.

EXAMPLES

Additional embodiments are disclosed in further detail in the followingexamples, which are not in any way intended to limit the scope of theclaims.

Example 1 tert-Butyl4-[3-(benzo[d]thiazol-2-yl)-6,8,8-trimethyl-2-oxo-7,8-dihydro-2H-pyrano[3,2-g]quinolin-9(6H)-yl]butanoate(Compound I-1)

tert-Butyl4-(6-formyl-7-hydroxy-2,2,4-trimethyl-3,4-dihydroquinolin-1(2H)-yl)butanoate(0.19 g) was dissolved in ethanol (2 mL). Ethyl2-(benzo[d]thiazol-2-yl)acetate (0.124 g) was added and the mixture wasstirred at room temperature for 15 min. Piperidine (5 μL) was added andcolor of the reaction mixture turned to red-yellow. Reaction mixture wasleft stirring at room temperature overnight. Next day the crude reactionmixture underwent aqueous workup, drying and purification bychromatography (silica gel with petroleum ether/ethyl acetate as eluent)to afford Compound I-1. Purity, structure and composition were confirmedby HPLC, NMR and LCMS. MS (DUIS): MW Calculated 518.22. Found: (−) 517(M−1).

Example 24-[3-(Benzo[d]thiazol-2-yl)-6,8,8-trimethyl-2-oxo-7,8-dihydro-2H-pyrano[3,2-g]quinolin-9(6H)-yl]butanoicacid (Compound I-2)

Compound I-1 (51.8 mg) was dissolved in DCM (5 mL) and trifluoroaceticacid (0.5 mL) was added via syringe and the reaction mixture was leftstirring overnight at room temperature. Solvent was distilled off usingrotary evaporator, the residue triturated with water (5 mL) solidfiltered off and dried to afford Compound I-2. Purity, structure andcomposition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MWCalculated 462.16. Found: (−) 461 (M−1).

Example 3 tert-Butyl4-[3-(benzo[d]thiazol-2-yl)-6,8,8-trimethyl-2-oxo-2H-pyrano[3,2-g]quinolin-9(6H)-yl]butanoate(Compound I-3)

tert-Butyl4-(6-formyl-7-hydroxy-2,2,4-trimethyl-quinolin-1(2H)-yl)butanoate (0.19g) was dissolved in ethanol (2 mL). Ethyl2-(benzo[d]thiazol-2-yl)acetate (0.124 g,) was added and the mixture wasstirred at room temperature for 15 min. Piperidine (5 μL) was added andcolor of the reaction mixture turned to red-yellow. Reaction mixture wasleft stirring at room temperature overnight and the crude reactionmixture underwent aqueous workup, drying and purification bychromatography (silica gel with petroleum ether/ethyl acetate as eluent)to afford Compound I-3. Purity, structure and composition were confirmedby HPLC, NMR and LCMS. MS (DUIS): MW Calculated 516.22. Found: (−) 515(M−1); (+) 517 (M+1).

Example 44-[3-(Benzo[d]thiazol-2-yl)-6,8,8-trimethyl-2-oxo-2H-pyrano[3,2-g]quinolin-9(6H)-yl]butanoicacid (Compound I-4)

Compound I-3 (51.7 mg) was dissolved in DCM (6 mL) and trifluoroaceticacid (1 mL) was added via syringe and the reaction mixture was leftstirring overnight at room temperature. Solvent was distilled off usingrotary evaporator, and the residue was triturated with water (5 mL). Theformed solid was filtered off and dried to afford Compound I-4. Purity,structure and composition were confirmed by HPLC, NMR and LCMS. MS(DUIS): MW Calculated 460.15. Found: (+) 461 (M+1).

Example 5 T-Butyl4-[3-(benzoxazolyl-2-yl)-6,8,8-trimethyl-2-oxo-7,8-dihydro-2H-pyrano[3,2-g]quinolin-9(6H)-yl]butanoate(Compound I-5)

tert-Butyl4-(6-formyl-7-hydroxy-2,2,4-trimethyl-3,4-dihydroquinolin-1(2H)-yl)butanoate(0.18 g) was dissolved in ethanol (3 mL). Ethyl 2-(benzoxazolyl)acetate(0.124 g) was added and the mixture was stirred at room temperature for15 min. Piperidine (5 μL) was added. Color of the reaction mixtureturned to red-yellow. Reaction mixture was left stirring at roomtemperature overnight. The crude reaction mixture underwent aqueousworkup, drying and purification by chromatography (silica gel withpetroleum ether/ethyl acetate as eluent) to afford Compound 1-5. Purity,structure and composition were confirmed by HPLC, NMR and LCMS. MS(DUIS): MW Calculated 502.25. Found: (−) 501 (M−1).

Example 64-[3-Benzoxazol-2-yl)-6,8,8-trimethyl-2-oxo-7,8-dihydro-2H-pyrano[3,2-g]quinolin-9(6H)-yl]butanoicacid (Compound I-6)

Compound I-5 (50 mg) was dissolved in DCM (5 mL) and trifluoroaceticacid (0.5 mL) was added via syringe and the reaction mixture was leftstirring overnight at room temperature. Solvent was distilled off usingrotary evaporator, and the residue triturated with water (5 mL). Theformed solid was filtered off and dried to afford Compound I-6. Purity,structure and composition were confirmed by HPLC, NMR and LCMS. MS(DUIS): MW Calculated 446.16. Found: (−) 445 (M−1).

Example 7 Ethyl4-[3-(benzoxazol-2-yl)-6,8,8-trimethyl-2-oxo-7,8-2H-pyrano[3,2-g]quinolin-9(6H)-yl]butanoate(Compound I-7)

Ethyl4-[6-formyl-7-hydroxy-2,2,4-trimethyl-3,4-quinolin-1(2H)-yl]butanoate(0.17 g) was dissolved in anhydrous ethanol (2.5 mL). Ethyl2-(benzoxazol-2-yl)acetate (0.102 g) was added and the mixture wasstirred at room temperature for 15 min. Piperidine (5 μL) was added.Reaction mixture was left stirring at room temperature overnight. Thecrude reaction mixture underwent aqueous workup, drying and purificationby chromatography (silica gel with petroleum ether/ethyl acetate aseluent) to afford Compound I-7. Purity, structure and composition wereconfirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 472.20. Found:(+) 473 (M+1).

Example 84-[3-(Benzoxazol-2-yl)-6,8,8-trimethyl-2-oxo-2H-pyrano[3,2-g]quinolin-9(6H)-yl]butanoicacid (Compound I-8)

Compound I-7 (51.8 mg) was dissolved in acetic acid (2.5 mL) andhydrochloric acid (5 mL) was added and the reaction mixture was leftstirring overnight at 60° C. Solvent was distilled off using rotaryevaporator, and the residue was triturated with water (5 mL). CompoundI-8 was filtered off and dried in vacuum. Purity, structure andcomposition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MWCalculated 444.17. Found: (+) 445 (M+1).

Example 93-[4-(3-(benzoxazol-2-yl)-6,8,8-trimethyl-2-oxo-2H-pyrano[3,2-g]quinolin-9(8H)-yl)-N-(4-(tert-butoxy)-4-oxobutyl)butanamido]propane-1-sulfonicacid (Compound I-9)

Compound I-8 (50 mg, 112 μmol) was dissolved in dimethylformamide (1 mL)and then solvent distilled off in vacuo. This operation was repeated twomore times, then dried Compound I-8 was redissolved in DMF (1 mL) atroom temperature. (Benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate (PyBOP, 1.5 eq., 64 mg, 169 μmol) was added to theflask then N,N-Diisopropylethylamine (DIPEA, 3 eq., 336 μmol, 43 mg, 58μL) was added via micropipette. Reaction flask was sealed under nitrogengas. After reaction was completed, the activated dye solution in DMF wasmixed with 3-[(4-(tert-butoxy)-4-oxobutyl)amino]propane-1-sulfonate (1.5eq., 224 μmol, 63 mg). More DIPEA (3 eq., 336 μmol, 43 mg, 58 μL) wasadded. Flask was again sealed under nitrogen gas and left overnight atroom temperature. Reaction progress was monitored by TLC, HPLC and(LCMS). When reaction was complete, water (2 mL) was added, the reactionmixture was stirred for 15 min and then solvent was distilled off fromthe reaction mixture in vacuum at room temperature. Compound I-9 wasused in next step without any farther purification.

Example 104-[4-(3-(Benzoxazol-2-yl)-6,8,8-trimethyl-2-oxo-2H-pyrano[3,2-g]quinolin-9(8H)-yl)-N-(3-sulfopropyl)butanamido]butanoicacid (Compound I-10)

The dried crude compound I-9 was re-dissolved in dichloromethane (2 mL).Trifluoroacetic acid (0.5 mL) was added and the reaction was leftstirring overnight at room temperature. The reaction was quenched withwater concentrated in vacuum and then purified by preparative-HPLC.Purity, structure and composition of the product were confirmed by HPLC,NMR and LCMS. MS (DUIS): MW Calculated 651.23. Found: (−) 650 (M−1).

Example 114-[3-(Benzoxazol-2-yl)-8,8-dimethyl-2-oxo-6-(sulfomethyl)-2H-pyrano[3,2-g]quinolin-9(8H)-yl)butanoicacid (Compound I-11)

Sulfuric acid (2.5 mL) was cooled down to about 5° C. then compound I-8(45 mg) was added and the reaction mixture was stirred at 20-25° C. for1 h. Then reaction mixture was kept at room temperature overnight.Reaction mixture was diluted slowly with anhydrous ether in presence ofexternal cooling. Precipitate was filtered off, dissolved in a mixtureof water (5 mL) and acetonitrile (5 mL). Solution was filtered andpurified by preparative HPLC. Purity, structure and composition of theproduct were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated524.13. Found: (−) 623 (M−1).

Example 121-[(5-carboxypentyl)-4-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-10-yl]pyridiniumbromide (Compound I-12)

8-Hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde(0.217 g) was dissolved in ethanol (6 mL). To this solution1-[(5-carboxypentyl)-(4-etxoxycarbonyl)methylpyridinium bromide (0.36 g)was added and the mixture was stirred at room temperature for 15 min.Piperidine acetate solution prepared from piperidine (20 mg) and aceticacid (20 mg)/in ethanol (5 mL) was added and this reaction mixture wasstirred at 60° C. for 5 hours then stirred overnight at roomtemperature. Solvent was distilled off and the residue was dissolved ina mixture of water (15 mL) and acetonitrile (15 mL). The resultingsolution was filtered and purified by preparative HPLC. This compoundwas converted to more stable hydrobromide salt form to afford CompoundI-12. Purity, structure and composition were confirmed by HPLC, NMR andLCMS. MS (DUIS): MW Calculated 432.20. Found: (+) 433 (M+1).

Example 131-(5-Carboxypentyl)-4-(1,1,7,7-tetramethyl-11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-10-yl)pyridin-1-iumbromide (Compound I-13)

8-Hydroxy-2,3,6,7-tetrahydro-1,1,7,7-tetramethyl-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde(0.27 g) was dissolved in ethanol (7 mL).1-[(5-Carboxypentyl)-(4-etxoxycarbonyl)methylpyridinium bromide (0.36 g)was added and the mixture was stirred at room temperature for 30 min.Solution of piperidine acetate prepared from piperidine (20 mg) andacetic acid (20 mg) in ethanol (5 mL) was added. This reaction mixturewas stirred at 60° C. for 5 hours then stirred overnight at roomtemperature. The solvent was distilled off and the red-colored residuewas dissolved in a mixture of water (15 mL) and acetonitrile (15 mL).The resulting solution was filtered and purified by preparative HPLC.This compound was converted to the more stable hydrobromide salt form byreaction with HBr solution in acetic acid (10%, 0.3 mL) to affordCompound I-13. Purity, structure and composition were confirmed by HPLC,NMR and LCMS. MS (DUIS): MW Calculated 488.27. Found: (+) 489 (M+1).

Example 141-(5-Carboxypentyl)-4-(9-ethyl-2-oxo-6,7,8,9-tetrahydro-2H-pyrano[3,2-g]quinolin-3-yl)pyridin-1-iumbromide (Compound I-14)

1-Ethyl-7-hydroxy-1,2,3,4-tetrahydroquinoline-6-carbaldehyde (0.2 g) wasdissolved in ethanol (10 mL).1-[(5-Carboxypentyl)-(4-etxoxycarbonyl)methylpyridinium bromide (0.36 g)was added and the mixture was stirred at room temperature for 30 min.Solution of piperidine acetate prepared from piperidine (20 mg) andacetic acid (20 mg)/in ethanol (5 mL) was added. This reaction mixturewas stirred at 80° C. for 2 hours then stirred overnight at roomtemperature. The solvent was distilled off and the residue was dissolvedin a mixture of water (10 mL) and acetonitrile (10 mL). The resultingsolution was filtered and purified by preparative HPLC. This compoundwas converted to the more stable hydrobromide salt form by reaction withHBr solution in acetic acid (10%, 0.3 mL) to afford Compound I-14.Purity, structure and composition were confirmed by HPLC, NMR and LCMS.MS (DUIS): MW Calculated 420.20. Found: (+) 421 (M+1).

Example 154-[4-(9-(4-(tert-Butoxy)-4-oxobutyl)-6,8,8-trimethyl-2-oxo-6,7,8,9-tetrahydro-2H-pyrano[3,2-g]quinolin-3-yl)pyridin-1-ium-1-yl]butane-1-sulfonate(Compound I-15)

tert-Butyl4-(6-formyl-7-hydroxy-2,2,4-trimethyl-3,4-dihydroquinolin-1(2H)-yl)butanoate(0.72 g) was dissolved in ethanol (25 mL).4-[4-(2-Ethoxy-2-oxoethyl)pyridin-1-ium-1-yl)butane-1-sulfonate (0.60 g)was added and the mixture was stirred at room temperature for 30 min.Solution of piperidine acetate prepared from piperidine (50 mg) andacetic acid (50 mg)/in ethanol (15 mL) was added. This reaction mixturewas stirred at 80° C. for 3 hours then stirred overnight at roomtemperature. The solvent was distilled off and the residue was dissolvedin a mixture of water (20 mL) and acetonitrile (20 mL). The resultingsolution was filtered and purified by preparative HPLC to affordCompound I-15. Purity, structure and composition were confirmed by HPLC,NMR and LCMS. MS (DUIS): MW Calculated 598.20. Found: (+) 599 (M+1).

Example 164-[4-(9-(3-Carboxypropyl)-6,8,8-trimethyl-2-oxo-6,7,8,9-tetrahydro-2H-pyrano[3,2-g]quinolin-3-yl)pyridin-1-ium-1-yl]butane-1-sulfonate(Compound I-16)

Compound I-15 was dissolved in DCM (5 ml) and TFA was added. Reactionmixture was stirred overnight at room temperature. Solvent was distilledoff in vacuum. To the residue water (1.5 ml) and acetonitrile (10 mL)were added and the solvent was distilled off again to remove TFA. Theremaining orange crystalline product was dissolved in a mixture of water(12 mL) and acetonitrile (12 mL). The resulting solution was filteredand purified by preparative HPLC. Purity, structure and composition wereconfirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 542.21. Found:(+) 543 (M+1).

Example 174-[4-(9-(4-(tert-Butoxy)-4-oxobutyl)-6,8,8-trimethyl-2-oxo-8,9-dihydro-2H-pyrano[3,2-g]quinolin-3-yl)pyridin-1-ium-1-yl]butane-1-sulfonate(Compound I-17)

tert-Butyl4-(6-formyl-7-hydroxy-2,2,4-trimethyl-quinolin-1(2H)-yl)butanoate (0.36g) was suspended in ethanol (5 mL).4-[4-(2-Ethoxy-2-oxoethyl)pyridin-1-ium-1-yl)butane-1-sulfonate (0.30 g)was added and the mixture was stirred at room temperature for 30 min.Solution of piperidine acetate prepared from piperidine (50 mg) andacetic acid (50 mg) in ethanol (15 mL) was added. This reaction mixturewas stirred at 50° C. for 5 hours then stirred overnight at roomtemperature. Solvent was distilled off and the residue was dissolved ina mixture of water (10 mL) and acetonitrile (10 mL). The resultingsolution was filtered and purified by preparative HPLC to affordCompound I-17. Purity, structure and composition were confirmed by HPLC,NMR and LCMS. MS (DUIS): MW Calculated 596.26. Found: (+) 597 (M+1).

Example 184-[4-(9-(3-Carboxypropyl)-6,8,8-trimethyl-2-oxo-8,9-dihydro-2H-pyrano[3,2-g]quinolin-3-yl)pyridin-1-ium-1-yl]butane-1-sulfonate(Compound I-18)

Compound I-17 was dissolved in DCM (5 ml) and TFA was added. Thereaction mixture was stirred overnight at room temperature then solventwas distilled off in vacuum. To the residue water (2 ml) andacetonitrile (10 mL) were added and solvent distilled off again toremove the residue TFA. The remaining orange crystalline product wastriturated with ether, filtered off and purified by preparative HPLC toafford Compound I-18. Purity, structure and composition were confirmedby HPLC, NMR and LCMS. MS (DUIS): MW Calculated 540.19. Found: (−) 539(M−1); (+) 541 (M+1).

Example 194-(4-(9-(4-((4-(tert-Butoxy)-4-oxobutyl)(3-sulfopropyl)amino)-4-oxobutyl)-6,8,8-trimethyl-2-oxo-8,9-dihydro-2H-pyrano[3,2-g]quinolin-3-yl)pyridin-1-ium-1-yl)butane-1-sulfonate(Compound I-19)

Compound I-8 (50 mg) was dissolved in DMF (1 mL) and then solventdistilled off in vacuo. This operation was repeated twice. The driedCompound I-8 was re-dissolved in DMF (1.5 mL) at room temperature.(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate(PyBOP, 1.5 eq., 64 mg, 169 μmol) was added to the flask then excess ofDIPEA (60 μL) was added. The reaction flask was sealed under nitrogengas. After reaction was complete, the activated dye solution in DMF wasmixed with 3-[(4-(tert-butoxy)-4-oxobutyl)amino]propane-1-sulfonate (63mg). More DIPEA (58 μL) was added. Flask was again sealed under nitrogengas and left overnight at room temperature. Reaction progress wasmonitored by TLC, HPLC and (LCMS). When reaction was complete, water (2mL) was added. The reaction mixture was stirred for 15 min and thensolvent distilled off from the reaction mixture in vacuum at roomtemperature to afford Compound I-19. This compound was used in next stepwithout any further purification.

Example 204-(4-(9-(4-((3-Carboxypropyl)(3-sulfopropyl)amino)-4-oxobutyl)-6,8,8-trimethyl-2-oxo-8,9-dihydro-2H-pyrano[3,2-g]quinolin-3-yl)pyridin-1-ium-1-yl)butane-1-sulfonate(Compound I-20)

The dried compound I-19 was dissolved in dichloromethane (5 mL).Trifluoroacetic acid (0.5 mL) was added and the reaction was leftstirring overnight at room temperature. The reaction was quenched withwater (0.5 mL), concentrated in vacuo and then purified bypreparative-HPLC. Purity, structure and composition of the product wereconfirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 747.25. Found:(−) 746 (M−1).

Example 211-(5-Carboxypentyl)-4-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)pyridin-1-iumbromide (Compound I-21)

4-Diethylaminosalicilic aldehyde (0.19 g) was dissolved in ethanol (5mL). 1-[(5-Carboxypentyl)-(4-etxoxycarbonyl)methylpyridinium bromide(0.36 g) was added and the mixture was stirred at room temperature for30 min. Solution of piperidine acetate prepared from piperidine (10 mg)and acetic acid (10 mg)/in ethanol (5 mL) was added. This reactionmixture was stirred at 70° C. for 5 hours then stirred overnight at roomtemperature. The product was filtered off, suspended in HBr solution inacetic acid (20%, 1 mL). This suspension was stirred at room temperaturefor 2 hours and the final product was filtered off to afford the morestable HBr salt form Compound I-21. This dye was used in next stepwithout further purification. Purity, structure and composition wereconfirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 408.20. Found:(+) 409 (M+1), 817 (2M+1).

Table 2 summarizes the yield, characterization data, and spectralproperties of the new coumarin fluorescent dyes disclosed in theexamples.

TABLE 2 Spectral properties Yield Absorption Fluorescence Stokes ShiftCompound # (%) nm nm nm I-1 65 n/a n/a n/a I-2 78 469 (CH₃OH) 512(CH₃OH) 43 I-3 84 n/a n/a n/a I-4 87 475 (H₂O) n/a n/a I-5 39 465 (EtOH)500 (EtOH) 35 I-6 54 459 (EtOH) 498 (EtOH) 39 463 (H₂O) I-7 45 465(EtOH) n/a n/a I-8 76 470 (EtOH) 515 (EtOH) 45 I-9 77 n/a n/a n/a I-1087 470 (H₂O) 521 (H₂O) 51 I-11 45 470 (H₂O) n/a n/a I-12 56 502 (H₂O)n/a n/a I-13 69 500 (H₂O) n/a n/a I-14 77 495 (EtOH) 565 (EtOH) 70 I-1559 500 (EtOH) n/a n/a I-16 76 501 (EtOH) 575 (EtOH) 74 I-17 78 510(EtOH) n/a n/a I-18 79 509 (EtOH) 592 (EtOH) 83 I-19 53 512 (EtOH) n/an/a I-20 58 510 (EtOH) n/a n/a I-21 76 482 (EtOH) 555 (EtOH) 73

Example 22 General Procedure for the Synthesis of Fully FunctionalNucleotide Conjugates

The coumarin fluorescent dyes disclosed herein is coupled withappropriate amino-substituted nucleotide A-LN3-NH₂ or C-LN3-NH₂ afteractivation of carboxylic group:

The dye (10 μmol) is dissolved in dimethylformamide (1 mL) and thensolvent is distilled off in vacuo. This procedure is repeated two moretimes. The dried dye is dissolved in N,N-dimethylacetamide (DMA, 0.2 mL)in a 5 mLround-bottomed flask at room temperature.

N,N,N′,N′-Tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate (TSTU,1.5 eq., 15 μmol, 4.5 mg) is added to the flask thanN,N-diisopropylethylamine (DIPEA, 3 eq., 30 μmol, 3.8 mg, 5.2 μL) isadded via micropipette to this solution. Reaction flask is sealed undernitrogen gas. After 15 minutes, the reaction progress is monitored byTLC (eluent H₂O/Acetonitrile 1:9) and HPLC. Meanwhile, solution ofappropriate N-LN3-NH₂ derivatives (20 mM, 1.5 eq, 15 μmol, 0.75 mL) isconcentrated in vacuo then re-dissolved in water (20 μL). Solution ofthe activated dye in DMA is transferred to the flask containing thesolution of N-LN3-NH₂. More DIPEA (3 eq, 30 μmol, 3.8 mg, 5.2 μL) isadded along with triethylamine (1 μL) Progress of coupling is monitoredhourly by TLC, HPLC and LCMS.

When reaction is complete, triethylamine bicarbonate buffer (TEAB, 0.05Mapprox., 3 mL) is added via pipette. Initial purification of the fullyfunctionalized nucleotide is carried out by running the quenchedreaction mixture through a DEAE-Sephadex® column (Sephadex poured intoan empty 25 g Biotage cartridge, solvent system TEAB/MeCN). This removesmost remaining dye.

Fractions from the Sephadex column is concentrated in vacuo. The crudematerial is re-dissolved in the minimum volume of water andacetonitrile, before filtering through a 20 μm Nylon filter. Thefiltered solution is purified by preparative-HPLC. Composition ofprepared compounds was confirmed by LCMS.

Table 3 summarizes the structure and spectral properties of variousnucleotides labelled with new coumarin dyes disclosed herein.ffA-LN3-Dye refers to a fully functionalized A nucleotide with LN3linker and labeled with a coumarin dye disclosed herein. ffC-LN3-Dyerefers to a fully functionalized C nucleotide with LN3 linker andlabeled with a coumarin dye disclosed herein. The R group in each of thestructures refers to the coumarin dye moiety after conjugation.

TABLE 3 Spectral properties Absorption Fluorescence Stokes Shift Compd.R nm nm nm C-I-1

488 (Tris) 524 (Tris) 36 A-I-4

499 (Tris) 538 (Tris) 39 C-I-6

473 (USM) 507 (USM) 34 C-I-8

455 (Tris) 524 (Tris) 69 C-I-10

480 (Tris) 524 (Tris) 43 A-I-13

519 (Tris) 579 (Tris) 80 A-I-14

513 (SRE) 573 (SRE) 60 A-I-16

504 (SRE) 570 (SRE) 66 A-I-18

511 (Tris) 593 (Tris) 82 A-I-20

514 (H₂O) 593 (SRE) 78 A-DY510XL (reference)

493 (H₂O) 585 (H₂O) 92

The efficiency of the A nucleotides labelled with the new coumarin dyeI-16 with long Stokes Shift was demonstrated by comparison withappropriate A nucleotides labelled with commercial dyes DY510XL andChromeo™ 494 (CH494). In this sequencing example, the two-channeldetection method was used. With respect to the two-channel methodsdescribed herein, nucleic acids can be sequenced utilizing methods andsystems described in U.S. Patent Application Publication No.2013/0079232, the disclosure of which is incorporated herein byreference in its entirety. In the two-channel detection, a nucleic acidcan be sequenced by providing a first nucleotide type that is detectedin a first channel, a second nucleotide type that is detected in asecond channel, a third nucleotide type that is detected in both thefirst and the second channel and a fourth nucleotide type that lacks alabel that is not, or minimally, detected in either channel. Thescatterplots were generated by RTA2.0.93 analysis of an experiment tocompare the relative intensities of fully functionalized A nucleotidelabeled with I-16, Dy510XL and Chromeo™ 494. The comparisons were madein the same run (same flow cell and sequencing reagents) byrehybridizing the sequencing primer and performing a short (26 cycles)SBS run. The scatterplots illustrated in FIG. 1 through FIG. 3 were atcycle 5 of each of the 26 cycle runs.

FIG. 1 illustrates the scatterplot of a fully functionalized nucleotide(ffN) mixture containing: A-I-16 (2 μM), C—NR440 (2 μM), dark G (2 μM)and T-NR550S0 (1 μM) in incorporation buffer with Pol812 (Blue exposure(Chan 1) 500 ms, Green exposure (Chan 2) 1000 ms; Scanned in Scanningmix).

FIG. 2 illustrates the scatterplot of a ffN mixture containing:A-DY510XL (2 μM), C—NR440 (2 μM), darkG (2 μM) and T-NR550S0 (1 μM) inincorporation buffer with Pol812 (Blue exposure (Chan 1) 500 ms, Greenexposure (Chan 2) 1000 ms; Scanned in SRE).

FIG. 3 illustrates the scatterplot of a ffN mixture containing: A-CH494(2 μM), C—NR440 (2 μM), darkG (2 μM) and T-NR550S0 (1 μM) inincorporation buffer with Pol812 (Blue exposure (Chan 1) 500 ms, Greenexposure (Chan 2) 1000 ms; Scanned in Scanning mix).

In each of FIGS. 1-3, “G” nucleotide is unlabeled and shown as the lowerleft cloud (“dark G”). The signal from the new coumarin dye I-16,DY510XL, and CH494 labeled “A” nucleotide is shown as the upper rightcloud in FIGS. 1, 2, and 3 respectively. The signal from the NR550S0 dyelabeled “T” nucleotide is indicated by the upper left cloud, and NR440dye labeled “C” nucleotide signal is indicated by the lower right cloud.The X-axis shows the signal intensity for one channel and the Y-axisshows the signal intensity for the other channel. It shows that thefully functional A-nucleotide conjugates labeled with dye I-16 providessufficient signal intensities and substantially better clouds separationas compared to commercial long Stokes Shift dyes DY510XL and CH494. Thestructure of NR440 is disclosed in U.S. provisional application No.62/402,635.

What is claimed is:
 1. A compound of Formula (I), or salts, mesomericforms thereof:

wherein R¹ is

and wherein R¹ is optionally substituted with one or more substituentsselected from the group consisting of alkyl, substituted alkyl, alkoxy,alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl,halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy,C-amido, N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonate,S-sulfonamido, N-sulfonamido, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted cycloalkyl, andoptionally substituted heterocyclyl; each R², R³, R⁴, R⁵, and R⁹ isindependently selected from the group consisting of H, alkyl,substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy,alkoxyalkyl, amino, aminoalkyl, halo, cyano, hydroxy, hydroxyalkyl,heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido, nitro, sulfonyl,sulfo, sulfino, sulfonate, S-sulfonamido, N-sulfonamido, optionallysubstituted carbocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl and optionally substituted heterocyclyl; each R⁶,R^(10a), R^(10b), and R^(10c) is independently selected from the groupconsisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl,haloalkyl, heteroalkyl, alkoxyalkyl, sulfonyl hydroxide, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted carbocyclyl, and optionally substituted heterocyclyl; eachR⁷ and R⁸ is independently selected from the group consisting of H,alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl,haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano, hydroxy,hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido,nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido,N-sulfonamido, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl and optionallysubstituted heterocyclyl; alternatively, R⁶ and R⁷ together with theatoms to which they are attached form a ring or ring system selectedfrom the group consisting of optionally substituted 5-10 memberedheteroaryl or optionally substituted 5-10 membered heterocyclyl; X isselected from the group consisting of O, S, NR¹¹, and Se; R¹¹ isselected from the group consisting of H, alkyl, substituted alkyl,alkenyl, alkynyl, aminoalkyl, carboxyalkyl, sulfonatoalkyl, haloalkyl,heteroalkyl, alkoxyalkyl, sulfo, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted carbocyclyl, andoptionally substituted heterocyclyl; and the bond represented by a solidand dashed line

is selected from the group consisting of a single bond and a doublebond, provided that when

is a double bond, then R³ is absent.
 2. The compound of claim 1, whereinR¹ is


3. The compound of claim 2, wherein X is O.
 4. The compound of claim 2,wherein X is S.
 5. The compound of claim 1, wherein R¹ is


6. The compound of claim 1, wherein R¹ is


7. The compound of claim 5 or 6, wherein each R^(10a) and R^(10b) is asubstituted alkyl.
 8. The compound of any one of claims 5 to 7, whereineach R^(10a) and R^(10b) is alkyl substituted with carboxyl,carboxylate, sulfo or sulfonate.
 9. The compound of any one of claims 1to 8, wherein R¹ is substituted with one or more substituents selectedfrom the group consisting of alkyl, halo, and C-carboxy.
 10. Thecompound of any one of claims 1 to 9, wherein the bond represented by asolid and dashed line

is a double bond.
 11. The compound of claim 10, wherein R² is alkyl. 12.The compound of claim 11, wherein R² is methyl.
 13. The compound of anyone of claims 1 to 12, wherein each R⁴ and R⁵ is H.
 14. The compound ofany one of claims 1 to 12, wherein at least one of R⁴ and R⁵ is alkyl.15. The compound of claim 14, wherein each R⁴ and R⁵ is alkyl.
 16. Thecompound of claim 15, wherein each R⁴ and R⁵ is methyl.
 17. The compoundof any one of claims 1 to 8, wherein the bond represented by a solid anddashed line

is a single bond.
 18. The compound of claim 17, wherein at least one ofR² and R³ is alkyl.
 19. The compound of claim 18, wherein each of R² andR³ is alkyl.
 20. The compound of claim 17, wherein at least one of R²and R³ is H.
 21. The compound of claim 20, wherein each of R² and R³ isH.
 22. The compound of any one of claims 17 to 21, wherein at least oneof R⁴ and R⁵ is H.
 23. The compound of any one of claims 17 to 21,wherein at least one of R⁴ and R⁵ is alkyl.
 24. The compound of any oneof claims 1 to 23, wherein R⁶ is a substituted alkyl.
 25. The compoundof claim 24, wherein R⁶ is alkyl substituted with carboxyl.
 26. Thecompound of claim 24, wherein R⁶ is alkyl substituted with —C(O)OR¹²,and wherein R¹² is selected from the group consisting of optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedheteroaryl, and optionally substituted 3 to 7 membered cycloalkyl. 27.The compound of claim 24, wherein R⁶ is alkyl substituted with—C(O)NR¹³R¹⁴, and wherein each R¹³ and R¹⁴ is independently selectedfrom H, optionally substituted alkyl, optionally substituted aryl,optionally substituted heteroaryl and optionally substituted 3 to 7membered cycloalkyl.
 28. The compound of claim 27, wherein each R¹³ andR¹⁴ is independently selected from alkyl substituted with one or moresubstituents selected from the group consisting of carboxyl,carboxylate, —C(O)OR¹², sulfo and sulfonate.
 29. The compound of any oneof claims 1 to 28, wherein R⁷ is H.
 30. The compound of any one ofclaims 1 to 23, wherein R⁶ and R⁷ are joined together with the atoms towhich they are attached to form an optionally substituted 6 memberedheterocyclyl.
 31. The compound of claim 30, wherein the optionallysubstituted 6 membered heterocyclyl contains one heteroatom.
 32. Thecompound of claim 30 or 31, wherein the 6 membered heterocyclyl issubstituted with one or more alkyl.
 33. The compound of any one ofclaims 1 to 32, wherein R⁸ is H.
 34. The compound of any one of claims 1to 33, wherein R⁹ is H.
 35. The compound of claim 1, selected from thegroup consisting of:

or salts, mesomeric forms thereof.
 36. The compound of any one of claims1 to 29, wherein the compound is covalently attached to a nucleotide oroligonucleotide via R⁶, and wherein R⁶ is a substituted alkyl.
 37. Thecompound of any one of claims 1 to 34, wherein the compound iscovalently attached to a nucleotide or oligonucleotide via R^(10a),R^(10b), or R^(10c), and wherein each of R^(10a), R^(10b), and R^(10c)is a substituted alkyl.
 38. A fluorescent compound of Formula (II), orsalts, mesomeric forms thereof with a Stokes shift at least about 60 nm:

wherein R^(Het) is a 5 to 10 membered heteroaryl optionally substitutedwith one or more R¹⁰; each R¹, R², R³, R⁴, and R⁵ is independentlyselected from the group consisting of H, alkyl, substituted alkyl,alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino,aminoalkyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy,O-carboxy, C-amido, N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonate,S-sulfonamido, N-sulfonamido, optionally substituted carbocyclyl,optionally substituted aryl, optionally substituted heteroaryl andoptionally substituted heterocyclyl; R⁶ is selected from the groupconsisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl,haloalkyl, heteroalkyl, alkoxyalkyl, sulfonyl hydroxide, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted carbocyclyl, and optionally substituted heterocyclyl; eachR⁷ and R⁸ is independently selected from the group consisting of H,alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl,haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano, hydroxy,hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido,nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido,N-sulfonamido, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl and optionallysubstituted heterocyclyl; alternatively, R⁶ and R⁷ together with theatoms to which they are attached form a ring or ring system selectedfrom the group consisting of optionally substituted 5-10 memberedheteroaryl or optionally substituted 5-10 membered heterocyclyl; eachR¹⁰ is independently selected from the group consisting of alkyl,substituted alkyl, alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl,alkoxyalkyl, sulfonyl hydroxide, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted carbocyclyl, andoptionally substituted heterocyclyl; the bond represented by a solid anddashed line

is selected from the group consisting of a single bond and a doublebond, provided that when

is a double bond, then R³ is absent.
 39. The fluorescent compound ofclaim 38, wherein R^(Het) is a 6 membered heteroaryl optionallysubstituted with one or more R¹⁰.
 40. The fluorescent compound of claim39, wherein R^(Het) is


41. The fluorescent compound of claim 38, wherein R^(Het) is a 9membered heteroaryl optionally substituted with one or more R¹⁰.
 42. Thefluorescent compound of claim 41, wherein R^(Het) is selected frombenzothiazolyl or benzoxazolyl, each optionally substituted with one ormore R¹⁰.
 43. The compound of any one of claims 38 to 42, wherein thebond represented by a solid and dashed line

is a double bond.
 44. The compound of claim 43, wherein R² is alkyl. 45.The compound of any one of claims 38 to 44, wherein at least one of R⁴and R⁵ is alkyl.
 46. The compound of any one of claims 38 to 42, whereinthe bond represented by a solid and dashed line

is a single bond.
 47. The compound of claim 46, wherein at least one ofR² and R³ is alkyl.
 48. The compound of claim 46, wherein at least oneof R² and R³ is H.
 49. The compound of any one of claims 46 to 48,wherein at least one of R⁴ and R⁵ is alkyl.
 50. The compound of any oneof claims 46 to 48, wherein at least one of R⁴ and R⁵ is H.
 51. Thecompound of any one of claims 38 to 50, wherein R⁶ is a substitutedalkyl.
 52. The compound of claim 51, wherein R⁶ is alkyl substitutedwith one or more substituents selected from carboxyl, —C(O)OR¹², and—C(O)NR¹³R¹⁴, wherein R¹² is selected from the group consisting ofoptionally substituted alkyl, optionally substituted aryl, optionallysubstituted heteroaryl, and optionally substituted 3 to 7 memberedcycloalkyl, and wherein each R¹³ and R¹⁴ is independently selected fromH, optionally substituted alkyl, optionally substituted aryl, optionallysubstituted heteroaryl and optionally substituted 3 to 7 memberedcycloalkyl.
 53. The compound of claim 52, wherein each R¹³ and R¹⁴ isindependently selected from alkyl substituted with one or moresubstituents selected from the group consisting of carboxyl,carboxylate, —C(O)OR¹², sulfo and sulfonate.
 54. The compound of any oneof claims 38 to 50, wherein R⁶ and R⁷ are joined together with the atomsto which they are attached to form an optionally substituted 6 memberedheterocyclyl.
 55. A nucleotide or oligonucleotide labeled with acompound according to any one of claims 1 to
 54. 56. The labelednucleotide or oligonucleotide of claim 55, wherein the compound iscovalently attached to the nucleotide or oligonucleotide via R⁶, andwherein R⁶ is a substituted alkyl.
 57. The labeled nucleotide oroligonucleotide of claim 55, wherein the compound is covalently attachedto the nucleotide or oligonucleotide via R¹⁰, and wherein R¹⁰ is asubstituted alkyl.
 58. The labeled nucleotide or oligonucleotide of anyone of claims 55 to 57, wherein the compound is attached to the C5position of a pyrimidine base or the C7 position of a 7-deaza purinebase of the nucleotide or oligonucleotide through a linker moiety. 59.The labeled nucleotide or oligonucleotide of any one of claims 55 to 58,further comprising a 3′-OH blocking group covalently attached to theribose or deoxyribose sugar of the nucleotide or oligonucleotide.
 60. Akit comprising one or more nucleotides wherein at least one nucleotideis a labeled nucleotide according to claims 55 to
 59. 61. The kit ofclaim 60, comprising two or more labeled nucleotides.
 62. The kit ofclaim 61, wherein two of the labeled nucleotides are excited using asingle laser.
 63. The kit of claim 61 or 62, further comprising a thirdand a fourth nucleotide, wherein each of the second, third and fourthnucleotide is labeled with a different compound, wherein each compoundhas a distinct absorbance maximum and each of the compound isdistinguishable from the other compounds.
 64. A method of sequencingcomprising incorporating a nucleotide according to any one of claims 55to 59 in a sequencing assay.
 65. The method of claim 64, furthercomprising detecting the nucleotide.
 66. The method of claim 64 or 65,wherein the sequencing assay is performed on an automated sequencinginstrument, and wherein the automated sequencing instrument comprisestwo light sources operating at different wavelengths.